Neuroprotective macrocyclic compounds and methods for their use

ABSTRACT

Embodiments of this invention provide novel peptidomimetics that contain a macrocycle. Such compounds are neuroprotective and have utility as therapeutic agents for treatment of diseases, injuries and other conditions characterised by neuronal degeneration and/or death. Compounds are also useful for manufacture of medicaments useful for treatment of such conditions.

RELATED APPLICATION

This application claims priority to U.S. Provisional patent applicationsSer. No. 60/456,136, filed Mar. 20, 2003 and Ser. No. 60/505,119, filedSep. 23, 2003, both herein incorporated fully by reference.

FIELD OF THE INVENTION

The present invention relates to novel peptidomimetics containing amacrocycle and methods of their use. This invention also relates to theneuroprotective activity of such compounds. More particularly, thisinvention relates to the use of these compounds and pharmaceuticalcompositions thereof in the treatment of diseases and conditionscharacterised by neuronal degeneration and/or death.

BACKGROUND

The degeneration and/or death of cells in the nervous system is a majorfactor in many diseases and medical conditions. Such diseases andconditions include traumatic brain injuries, traumatic spinal cordinjuries, stroke, hypoxia or ischemia related to decreased neuralperfusion secondary to cardiac arterial bypass graft surgery (CABG),Parkinson's disease, Alzheimer's disease, multiple sclerosis,amyotrophic lateral sclerosis and other neurodegenerative diseases. Itis of interest to prevent or decrease such cell death and degenerationand to minimize the loss of neural function.

Certain compounds are useful as neuroprotective agents. One suchcompound is insulin-like growth factor 1 (IGF-1) (Scheepens et al,WO00/13650). IGF-1 is a naturally occurring peptide that can decreasethe binding of glutamate to the glutamate receptors of neurons(Bourguinon, U.S. Pat. No. 5,804,550). IGF-1 can also decrease neuronaldegradation caused by damage and disease. IGF-1 is cleaved byproteolysis in vivo to give des₁₋₃ IFG-1 and the N-terminal tripeptideGly-Pro-Glu (GPE). GPE and analogues have been found to beneuroprotective (Gluckman et al, U.S. Pat. No. 6,187,906 incorporatedherein by reference).

However, such peptides may not be ideal for the treatment of neuraldeath and degeneration especially in conditions in which they arerapidly metabolised in vivo. There is a need for compounds that haveneuroprotective and neuroregenerative properties and that are moremetabolically stable, especially to enzymatic degradation.

The use of peptidomimetics to mimic the behaviour of biologically activepeptides is common in the pursuit of a drug candidate. Peptidomimeticsthat are more metabolically stable and protease resistant than peptidesare desirable, and they often adopt well-defined conformations. Cyclicstructures can provide a rigid geometry that can be used to probe thebioactive conformation of a given peptide. This rigidityconformationally restricts the molecule and thus provides a method todesign molecules with enhanced biological activity.

SUMMARY

Aspects of this invention include novel cyclic peptidomimetics that canmimic certain properties of the tripeptide Gly-Pro-Glu (GPE). Certainaspects of the invention include molecules having the structuralformulae and substituents described below:

In certain embodiments, compounds of Formula 1 and Formula 2 includesubstituents where:

R¹ and R² are independently selected from the group consisting of —OR′,—SR′, —NR′R′, —NO₂, —CN, —C(O)R′, —C(O)OR′, —C(O)NR′R′, —C(NR′)NR′R′,trihalomethyl, halogen, alkyl, substituted alkyl, heteroalkyl,substituted heteroalkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, arylalkyl, substituted arylalkyl, heteroarylalkyl andsubstituted heteroarylalkyl; each R′ is independently selected from thegroup consisting of —H, alkyl, heteroalkyl, alkenyl, alkynyl, aryl,arylalkyl, heteroaryl and heteroarylalkyl and;

R³ is selected from the group consisting of —H, —OR′, —SR′, —NR′R′,—NO₂, —CN, —C(O)R′, —C(O)OR′, —C(O)NR′R′, —C(NR)NR′R′, trihalomethyl,halogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, arylalkyl,substituted arylalkyl, heteroarylalkyl and substituted heteroarylalkyl;each R′ is independently selected from the group consisting of —H,alkyl, heteroalkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl andheteroarylalkyl and;

X is a linear alkyl or alkenyl chain of C₀-C₃.

In other embodiments, compounds of Formula 3 and Formula 4 includesubstituents where:

R¹ and R² are independently selected from the group consisting of —H,—OR′, —SR′, —NR′R′, —NO₂, —CN, —C(O)R′, —C(O)OR′, —C(O)NR′R′,—C(NR′)NR′R′, trihalomethyl, halogen, alkyl, substituted alkyl,heteroalkyl, substituted heteroalkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, arylalkyl, substituted arylalkyl,heteroarylalkyl and substituted heteroarylalkyl; each R′ isindependently selected from the group consisting of —H, alkyl,heteroalkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl andheteroarylalkyl and;

X is a linear alkyl or alkenyl chain of C₀-C₄.

In other embodiments, compounds of Formula 5 and Formula 6 includesubstituents where:

R is independently selected from the group consisting of —H, —OR′, —SR′,—NR′R′, —NO₂, —CN, —C(O)R′, —C(O)OR′, —C(O)NR′R′, —C(NR′)NR′R′,trihalomethyl, halogen, alkyl, substituted alkyl, heteroalkyl,substituted heteroalkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, arylalkyl, substituted arylalkyl, heteroarylalkyl andsubstituted heteroarylalkyl; each R′ is independently selected from thegroup consisting of —H, alkyl, heteroalkyl, alkenyl, alkynyl, aryl,arylalkyl, heteroaryl and heteroarylalkyl and;

X is a linear alkyl or alkenyl chain of C₀-C₄.

In other embodiments, compounds of Formula 7 and Formula 8 includesubstituents where:

R is independently selected from the group consisting of —H, —OR′, —SR′,—NR′R′, —NO₂, —CN, —C(O)R′, —C(O)OR′, —C(O)NR′R′, —C(NR′)NR′R′,trihalomethyl, halogen, alkyl, substituted alkyl, heteroalkyl,substituted heteroalkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, arylalkyl, substituted arylalkyl, heteroarylalkyl andsubstituted heteroarylalkyl; each R′ is independently selected from thegroup consisting of —H, alkyl, heteroalkyl, alkenyl, alkynyl, aryl,arylalkyl, heteroaryl and heteroarylalkyl.

Other aspects of the invention provide pharmaceutically acceptable saltsof the compounds described in formulas 1-8.

In still other aspects, this invention provides pharmaceuticalcompositions comprising a pharmaceutically acceptable excipient and atherapeutically effective amount of at least one compound of thisinvention. These compositions find use as anti-apoptotic andanti-necrotic agents and for other conditions involving neuraldegeneration or injury.

In yet further aspects, this invention provides a method of treating ananimal having a disease or injury capable of treatment by administrationof a suitable compound, comprising administration to that animal of atleast one compound of this invention, optionally in conjunction with atleast one other conventional therapeutic agent for the disease beingtreated.

In still further aspects the animal to be treated is a human.

In still further aspects, this invention provides methods of preparingthe compounds of Formulas 1-8 of this invention.

In yet other aspects, this invention provides methods of synthesising,formulating and preparing pharmaceutical preparations comprisingcompounds of Formulas 1-8 of this invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing effects of(2S,3′S,8′R,11′S)2-{[(3′-Amino-1′-aza-2′-oxobicyclo[6.3.0]-undecyl)-11′-carbonyl]amino}-1,5-pentanedioicacid trifluoroacetate salt 48 on neuronal survival in animals followingexcitotoxic oxidative stress.

FIG. 2 is a graph showing effects of(2S,9′R,12′S)-2-{[(1′,4′-Diaza-2′-oxobicyclo[7.3.0]dodecyl)-12′-carbonyl]amino}-1,5-pentanedioicacid trifluoroacetate 68 on neuronal survival in animals followingexcitotoxic oxidative stress.

FIG. 3 is a graph showing the neuroprotective effects of(2S,9′R,12′S)-2-{[(1′,4′-Diaza-2′-oxobicyclo[7.3.0]dodecyl)-12′-carbonyl]amino}-1,5-pentanedioicacid trifluoroacetate 68 in a global model of brain ischaemia.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

“Alkenyl” refers to an unsaturated branched, straight chain or cyclichydrocarbon radical having at least one carbon-carbon double bond. Theradical can be in either the cis or trans conformation about the doublebond(s). Exemplary alkenyl groups include allyl, ethenyl, propenyl,isopropenyl, butenyl, isobutenyl, cyclopentenyl and the like. In someembodiments the alkenyl groups are (C₂-C₆) alkenyl and in otherembodiments allyl can be particularly useful.

“Alkyl” refers to a saturated branched, straight chain or cyclichydrocarbon radical. Exemplary alkyl groups include methyl, ethyl,isopropyl, cyclopropyl, tert-butyl, cyclopropylmethyl, hexyl and thelike. In some embodiments the alkyl groups are (C₁-C₆) alkyl.

“Alkynyl” refers to an unsaturated branched, straight chain or cyclichydrocarbon radical having at least one carbon-carbon triple bond.Exemplary alkynyl groups include ethynyl, propynyl, butynyl, isobutynyland the like. In some embodiments the alkynyl group is (C₂-C₆) alkynyl.

“Aryl” refers to an unsaturated cyclic hydrocarbon radical with aconjugated π electron system. Exemplary aryl groups include phenyl,naphthyl and the like. In some embodiments the aryl group is (C₅-C₂₀)aryl.

“Arylalkyl” refers to a straight chain alkyl, alkenyl or alkynyl groupwherein one of the hydrogen atoms bonded to the terminal carbon isreplaced with an aryl group. Exemplary arylalkyl groups include benzyl,naphthylmethyl, benzylidene and the like.

A “growth factor” refers to an extracellular polypeptide-signalingmolecule that stimulates a cell to grow or proliferate.

“Heteroalkyl” refers to an alkyl moiety wherein one or more carbon atomsare replaced with another atom such as N, P, O, S etc. Exemplaryheteroalkyl groups include pyrrolidine, morpholine, piperidine,piperazine, imidazolidine, pyrazolidine, terahydrofuran, (C₁-C₁₀)substituted amines, (C₂-C₆) thioethers and the like.

“Heteroaryl” refers to an aryl moiety wherein one or more carbon atomsare replaced with another atom such as N, P, O, S etc. Exemplaryheteroaryl groups include carbazole, furan, imidazole, indazole, indole,isoquinoline, purine, pyrazine, pyrazole, pyridazine, pyridine, pyrrole,thiazole, thiophene, triazole and the like.

“Injury” includes any acute or chronic damage of an animal that resultsin degeneration or death of cells in the nervous system or results inloss of function. Such cells include neuronal cells and non-neuronalcells. Injury includes stroke, non-hemorrhagic stroke, traumatic braininjury, perinatal asphyxia associated with fetal distress such asfollowing abruption, cord occlusion or associated with intrauterinegrowth retardation, perinatal asphyxia associated with failure ofadequate resuscitation or respiration, severe CNS insults associatedwith near miss drowning, near miss cot death, carbon monoxideinhalation, ammonia or other gaseous intoxication, cardiac arrest, coma,meningitis, hypoglycemia and status epilepticus, episodes of cerebralasphyxia associated with coronary bypass surgery, hypotensive episodesand hypertensive crises, and cerebral trauma. It is to be understoodthat the above examples are by way of illustration only, and are notintended to be a complete listing of injuries capable of being treatedby the compounds and methods of this invention.

A “pharmaceutically acceptable excipient” refers to an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients can be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

A “pharmaceutically acceptable salt” refers to a salt that ispharmaceutically acceptable and has the desired pharmacologicalproperties. Such salts include salts that can be formed where acidicprotons present in the compounds are capable of reacting with inorganicor organic bases. Suitable inorganic salts include those formed with thealkali metals, e.g. sodium and potassium, magnesium, calcium, andaluminium. Suitable organic salts include those formed with organicbases such as the amine bases e.g. ethanolamine, diethanolamine,triethanolamine, tromethamine, N-methylglucamine, and the like. Suchsalts also include acid addition salts formed with inorganic acids (e.g.hydrochloric and hydrobromic acids) and organic acids (e.g. acetic acid,citric acid, maleic acid, and the alkane- and arene-sulfonic acids suchas methanesulfonic acid and benzenesulfonic acid). When there are twoacidic groups present, a pharmaceutically acceptable salt can be amono-acid mono-salt or a di-salt; and similarly where there are morethan two acidic groups present, some or all of such groups can besalified.

A “protecting group” has the meaning conventionally associated with itin organic synthesis, i.e. a group that selectively blocks one or morereactive sites in a multifunctional compound such that a chemicalreaction can be carried out selectively on another unprotected reactivesite and such that the group can readily be removed after the selectivereaction is complete.

“Substituted” refers to where one or more of the hydrogen atoms on analkyl, heteroalkyl, alkenyl, alkynyl, aryl, heteroaryl or arylalkylradical are independently replaced with another substituent.Substituents include —R′, —OR′, —SR′, —NR′R′, —NO₂, —CN, —C(O)R′,—C(O)OR′, —C(O)NR′R′, —C(NR′)NR′R′, —NR′—C(NR′)—OR′, —NR′—C(NR′)—SR′,NR′—C(NR′)—NR′R′, trihalomethyl and halogen where each R′ isindependently —H, alkyl, heteroalkyl, alkenyl, alkynyl, aryl, arylalkyl,heteroaryl or heteroarylalkyl.

A “therapeutically effective amount” means the amount that, whenadministered to an animal for treating a disease, is sufficient toeffect treatment for that disease; that is, an amount that decreasesadverse symptoms or findings, promotes desirable symptoms or findings,and/or treats an underlying disorder, and/or is curative.

“Treating” or “treatment” of a disease includes preventing the diseasefrom occurring in an animal that may be predisposed to the disease butdoes not yet experience or exhibit symptoms of the disease (prophylactictreatment), inhibiting the disease (slowing or arresting itsdevelopment), providing relief from the symptoms or side-effects of thedisease (including palliative treatment), and relieving the disease(causing regression of the disease).

Implicit hydrogen atoms (such as the hydrogens on the pyrrole ring,etc.) are omitted from the formulae for clarity, but should beunderstood to be present.

Compounds of the Invention

Certain aspects of the invention provide molecules having the structuralformulae and substituents described below:

In certain embodiments, compounds of Formula 1 and Formula 2 includesubstituents where:

R¹ and R² are independently selected from the group consisting of —OR′,—SR′, —NR′R′, —NO₂, —CN, —C(O)R′, —C(O)OR′, —C(O)NR′R′, —C(NR′)NR′R′,trihalomethyl, halogen, alkyl, substituted alkyl, heteroalkyl,substituted heteroalkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, arylalkyl, substituted arylalkyl, heteroarylalkyl andsubstituted heteroarylalkyl; each R′ is independently selected from thegroup consisting of —H, alkyl, heteroalkyl, alkenyl, alkynyl, aryl,arylalkyl, heteroaryl and heteroarylalkyl and;

R³ is selected from the group consisting of —H, —OR′, —SR′, —NR′R′,—NO₂, —CN, —C(O)R′, —C(O)OR′, —C(O)NR′R′, —C(NR′)NR′R′, trihalomethyl,halogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, arylalkyl,substituted arylalkyl, heteroarylalkyl and substituted heteroarylalkyl;each R′ is independently selected from the group consisting of —H,alkyl, heteroalkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl andheteroarylalkyl and;

X is a linear alkyl or alkenyl chain of C₀-C₃.

In further embodiments of the invention the compounds are compounds ofFormula 1 where R¹═—COOH; R²═—(CH₂)₂—COOH; R³═H; X═—(CH₂)₃—

In other embodiments of the invention the compounds are compounds ofFormula 1 where R¹═—COOH; R²═—(CH₂)₂—COOH; R³═—CH₃; X═—(CH₂)₃—

In still other embodiments of the invention the compounds are compoundsof formula 1 where R¹═—COOH; R²═—(CH₂)₂—COOH; R³═allyl; X═—(CH₂)₃—

In yet further embodiments of the invention the compounds are compoundsof formula 1 where R¹═—COOH; R²═—(CH₂)₂—CH₃; R³═H; X═—(CH₂)₃—

In still other embodiments of the invention the compounds are compoundsof formula 1 where R¹═—COOH; R²═—(CH₂)₂—COOH; R³═H; X═—(CH₂)₂—

In further embodiments of the invention the compounds are compounds offormula 1 where R¹═—COOH; R²═—(CH₂)₂—COOH; R³═H; X═—CH₂—CH═CH—, (wherethe CH₂ of X is adjacent to the carbon attached to the NH₂ group).

In other embodiments, compounds of Formula 3 and Formula 4 includesubstituents where:

R¹ and R² are independently selected from the group consisting of —H,—OR′, —SR′, —NR′R′, —NO₂, —CN, —C(O)R′, —C(O)OR′, —C(O)NR′R′,—C(NR′)NR′R′, trihalomethyl, halogen, alkyl, substituted alkyl,heteroalkyl, substituted heteroalkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, arylalkyl, substituted arylalkyl,heteroarylalkyl and substituted heteroarylalkyl; each R′ isindependently selected from the group consisting of —H, alkyl,heteroalkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl andheteroarylalkyl and;

X is a linear alkyl or alkenyl chain of C₀-C₄.

In further embodiments of the invention the compounds are compounds offormula 3 where R¹═—CH(COOH)—(CH₂)₂—COOH; R²═H; X═—(CH₂)₄—

In still other embodiments of the invention the compounds are compoundsof formula 3 where R¹═—CH(COOH)—(CH₂)₂—COOH; R²═CH₃; X═—(CH₂)₄—

In yet further embodiments of the invention the compounds are compoundsof formula 3 where R¹═—CH(COOH)—(CH₂)₂—COOH; R²═allyl; X═—(CH₂)₄—

In further embodiments of the invention the compounds are compounds offormula 3 where R¹═—CH(COOH)—(CH₂)₂—CH₃; R²═H; X═—(CH₂)₄—

In other embodiments of the invention the compounds are compounds offormula 3 where R¹═—CH(COOH)—(CH₂)₂—COOH; R²═H; X═—(CH₂)₃—

In still other embodiments of the invention the compounds are compoundsof formula 3 where R¹═—CH(COOH)—(CH₂)₂—COOH; R²═H; X═—CH₂—CH═CH—CH₂—

In other embodiments, compounds of Formula 5 and Formula 6 includesubstituents where:

R is independently selected from the group consisting of —H, —OR′, —SR′,—NR′R′, —NO₂, —CN, —C(O)R′, —C(O)OR′, —C(O)NR′R′, —C(NR′)NR′R′,trihalomethyl, halogen, alkyl, substituted alkyl, heteroalkyl,substituted heteroalkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, arylalkyl, substituted arylalkyl, heteroarylalkyl andsubstituted heteroarylalkyl; each R′ is independently selected from thegroup consisting of —H, alkyl, heteroalkyl, alkenyl, alkynyl, aryl,arylalkyl, heteroaryl and heteroarylalkyl and;

X is a linear alkyl or alkenyl chain of C₀-C₄.

In other embodiments, compounds of Formula 7 and Formula 8 includesubstituents where:

R is independently selected from the group consisting of —H, —OR′, —SR′,—NR′R′, —NO₂, —CN, —C(O)R′, —C(O)OR′, —C(O)NR′R′, —C(NR′)NR′R′,trihalomethyl, halogen, alkyl, substituted alkyl, heteroalkyl,substituted heteroalkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, arylalkyl, substituted arylalkyl, heteroarylalkyl andsubstituted heteroarylalkyl; each R′ is independently selected from thegroup consisting of —H, alkyl, heteroalkyl, alkenyl, alkynyl, aryl,arylalkyl, heteroaryl and heteroarylalkyl.

In further embodiments of the invention the compounds are compounds offormula 7 where R═H.

In yet other embodiments of the invention the compounds are compounds offormula 7 where R═CH₃.

In yet further embodiment of the invention the compounds are compoundsof formula 7 where R=allyl.

Other aspects of the invention provide pharmaceutically acceptable saltsof the compounds described in Formulas 1-8.

In still other aspects, this invention provides pharmaceuticalcompositions comprising a pharmaceutically acceptable excipient and atherapeutically effective amount of at least one compound of thisinvention. These compositions find use as anti-apoptotic andanti-necrotic agents and for other conditions involving neuraldegeneration or injury.

In further aspects, this invention provides methods of treating ananimal having a disease or injury capable of treatment by administrationof a suitable compound of Formulas 1-8, comprising administration tothat animal of at least one compound of this invention, optionally inconjunction with at least one other conventional therapeutic agent forthe disease being treated.

In yet further aspects, the animal to be treated is a human.

In still further aspects, this invention provides methods ofsynthesizing, formulating and preparing pharmaceutical preparationscomprising compounds of Formulas 1-8 of this invention.

Those with skill in the art will appreciate that the above structuralformulae contain chiral centres, the number of which will depend on thedifferent substituents. The chirality is only indicated for somecentres. The chirality can be either R or S at each centre. The formulaedrawings represent only one of the possible tautomeric, conformationalisomeric or enantiomeric forms, it should be understood that theinvention encompasses any tautomeric, conformational isomeric orenantiomeric forms which exhibit biological or pharmacological activityas described herein.

Pharmacology and Utility

Certain aspects of this invention include the use of compounds of theinvention in treatment or prevention of cell damage, degeneration and/ordeath in mammals in response to injury or disease. Some embodimentscomprise delivering a composition containing a compound of the inventionto an animal suffering from neural degeneration, and in some cases,conditions involving apoptotic and necrotic cell death. In someembodiments, compositions are desirable to treat an injury or disease ofthe CNS affecting or liable to affect brain cells. Compositions areprovided that can also include one or more other agents that promoteneural regeneration, decrease cell degeneration or death, or areneuroprotective.

Such other agents can be selected from the group consisting of forexample, growth factors and associated derivatives, e.g., insulin-likegrowth factor-I [IGF-I], insulin-like growth factor-II [IGF-II], thetripeptide GPE, transforming growth factor-β1, activin, growth hormone,nerve growth factor, growth hormone binding protein, and/or IGF-bindingproteins.

Other aspects of the invention include compositions and methods ofpromoting fasiculation of axons. By promoting formation of nervebundles, compounds of the invention can be useful in treating conditionsin which nerve processes (axons and/or dendrites) have become severed,such as in sharp force injuries, local areas of necrosis or disease, orother localized injuries to nerve processes.

In yet other embodiments, compositions and methods to treat or preventcell damage and death in response to injury and disease, including CNSinjury and disease, comprise administration of a therapeutic amount of acompound of the invention alone or in combination with other agents,after the insult. These embodiments can be particularly desirable insituations of unexpected injury, such as in cardiac arrest, trauma suchas head injuries caused by automobile accidents, head wounds and thelike.

In still further embodiments, compounds of the invention can be usedeither alone or in combination with other agents to prevent adverseeffects of planned brain injury. Such conditions include CABG or otherplanned surgeries such as brain surgery, vascular surgery or otherinterventions that can lead to decreased perfusion of the nervoussystem. By treating an animal, such as a human being, in advance and/orsimultaneously and/or after the surgery, adverse neurological effectsmay be ameliorated.

As indicated above, the present invention is, broadly based upon theapplicant's finding that compounds of the invention can protect cells,particularly nerve cells, against damage, loss of neurites, and/orapoptotic or necrotic cell death.

It is herein demonstrated that compounds of the invention exhibitneuroprotection in cell culture models of neurodegenerative disease andcan therefore be an effective addition or alternative to conventionaltherapies for neural degeneration.

Although the mechanism of the protective effects is not known, onepossible mechanism involves protecting cells from apoptotic and necroticcell death. However, regardless of the mechanism of action, compounds ofthe invention can be used as an effective therapy for a variety ofneurological diseases, including hypoxia, ischemia andneurotoxin-induced nerve damage. Moreover, compounds of the inventioncan be used in the absence of any particular neurological deficit topromote neurite outgrowth and fasiculation of nerves. Thus, insituations in which cell death is not necessarily associated with theneurological disorder (e.g., axonal damage such as caused by spinal cordinjury), administration of compounds of the invention may be aneffective way of promoting neurite regeneration.

Therapeutic Applications

Compositions and methods of the invention find use in the treatment ofanimals, such as human patients, suffering from neural injury ordisease. Still more generally, the compositions and methods of theinvention find use in the treatment of mammals, such as human patients,suffering from nerve damage or potential apoptotic and/or necrotic celldeath, due to injuries and diseases.

Specific conditions and diseases characterised by neuronal degeneration,apoptosis and/or necrosis include but are not limited to Alzheimer'sdisease, Parkinson's disease, multiple sclerosis, amyotrophic lateralsclerosis, spinal muscular atrophy, peripheral neuropathy,Creutzfeldt-Jakob disease, AIDS dementia, progressive supranuclearpalsy, myelinopathia centralis diffusa (vanishing white matter disease),chronic neurodegenerative disease, Huntington's disease, stroke,ischemic injury, hypoxic injury, reperfusion injury, head injury, CNStrauma, epilepsy, cerebral ischemia, glaucoma, retinal disorders, opticneuropathy, optic neuritis, Down's syndrome, encephalomyelitis,meningitis, panencephalitis, neuroblastoma, schizophrenia anddepression. Each of the above conditions exhibits pathophysiologicalfindings and symptoms that are mimicked by neurotoxicity associated withglutamate toxicity.

Still more generally, the invention has application in the induction ofnerve bundle formation following insult in the form of trauma, toxinexposure, asphyxia or hypoxia-ischemia. Additionally, the invention hasapplication in the treatment or prevention of apoptosis in response toinjury or disease in the form of cancers, viral infections, autoimmunediseases, neurological diseases and injuries and cardiovasculardiseases.

Treatment can be given before an injury, for example, before electivesurgery. Examples of relevant elective procedures include neuralsurgery, in which retraction of lobes of the brain can lead to cerebraloedema, or heart operations, such as valve replacement, in whichinevitable small emboli are said to lead to detectable impairment ofbrain function in some 75% of cases.

Determining Efficacy

The anti-apoptotic and anti-necrotic activity of compounds of theinvention can be measured by in vivo using cell counts by methods knownto those skilled in the art including the methods of Klempt et al(Klempt et al, 1992, Molecular Brain Research: 13: 93-101), microscopicexaminations of morphology, cell counts of surviving and dead neuronsstained with thionin/fuchsin and the like. Compounds of the inventioncan also be measured in vitro using mass spectroscopy, immunological, orchromatographic methods known in the art.

CNS damage can for example be measured clinically by the degree ofpermanent neurological deficit cognitive function, and/or propensity toseizure disorders. Herein are disclosed histological techniques suitablefor measuring effects in vivo.

The therapeutic ratio of a compound is understood to be the ratio of (1)the mean dose that causes adverse side effect over (2) the mean dosethat causes a desirable therapeutic effect. Thus, for compounds forwhich have therapeutic effects at relatively low doses and undesirableside effects at high doses, the therapeutic ratio is >1. Therapeuticratio can be determined, for example, by comparing the dose thatproduces significant weight loss (or other observable side-effect)divided by the dose that produces anti-apoptotic and anti-necroticactivity in a suitable in vivo animal species such as the rat or mouse.Suitable models include a hypoxic-ischemic injury (Sirimanne et al, 1994Journal of Neuroscience Methods: 55: 7-14) and experimental immuneencephalomyelitis (Mendel et al., 1995 Eur. J. Immunol.: 25: 1951-1959).

Pharmaceutical Compositions and Administration

Compounds of the invention can be administered as part of a medicamentor pharmaceutical preparation. This can involve combining a compound ofthe invention with any pharmaceutically appropriate carrier, adjuvant orexcipient. The selection of the carrier, adjuvant or excipient will ofcourse usually be dependent upon the route of administration to beemployed.

In general, compounds of this invention will be administered intherapeutically effective amounts by any of the usual modes known in theart, either singly or in combination with other conventional therapeuticagents for the disease being treated. A therapeutically effective amountcan vary widely depending on the disease or injury, its severity, theage and relative health of the animal being treated, the potency of thecompound(s), and other factors. As anti-apoptotic and anti-necroticagents, therapeutically effective amounts of compounds of this inventioncan range from 0.001 to 100 milligrams per kilogram mass of the animal,with lower doses such as 0.001 to 0.1 mg/kg being appropriate foradministration through the cerebrospinal fluid, such as byintracerebroventricular administration, and higher doses such as 1 to100 mg/kg being appropriate for administration by methods such as oral,systemic (e.g. transdermal), or parenteral (e.g. intravenous)administration. A person of ordinary skill in the art will be ablewithout undue experimentation, having regard to that skill and thisdisclosure, to determine a therapeutically effective amount of acompound of this invention for a given disease or injury.

Compounds of the invention can be administered peripherally via anyperipheral route known in the art. These can include parenteral routesfor example injection into the peripheral circulation, subcutaneous,intraorbital, ophthalmic, intraspinal, intracisternal, topical, infusion(using e.g. slow release devices or minipumps such as osmotic pumps orskin patches), implant, aerosol, inhalation, scarification,intraperitoneal, intracapsular, intramuscular, intranasal, oral, buccal,transdermal, pulmonary, rectal or vaginal. The compositions can beformulated for parenteral administration to humans or other mammals intherapeutically effective amounts (e.g. amounts which eliminate orreduce the patient's pathological condition) to provide therapy for theneurological diseases described above.

Desirably, if possible, when administered as anti-apoptotic andanti-necrotic agents, compounds of this invention will be administeredorally. The amount of a compound of this invention in the compositioncan vary widely depending on the type of composition, size of a unitdosage, kind of excipients, and other factors well known to those ofordinary skill in the art. In general, the final composition cancomprise from 0.0001 percent by weight (% w) to 10% w of the compound ofthis invention, preferably 0.001% w to 1% w, with the remainder beingthe excipient or excipients.

Other convenient administration routes include subcutaneous injection(e.g. dissolved in a physiologically compatible carrier such as 0.9%sodium chloride) or direct administration to the CNS. Using stereotacticdevices and accurate maps of an animal's CNS, a compound can be injecteddirectly into a site of neural damage. Such routes of administration canbe especially desired in situations in which perfusion of that locationis compromised either by decreased vascular perfusion or by decreasedcerebral spinal fluid (CSF) flow to that area. Examples includeadministration by lateral cerebroventricular injection or through asurgically inserted shunt into the lateral cerebroventricle of the brainof the patient, intraveneously, direct injection into the desiredlocation or other routes.

The effective amount of compound in the CNS can be increased byadministration of a pro-drug form of a compound which comprises acompound of the invention and a carrier, where the carrier is joined toa compound of the invention by a linkage which is susceptible tocleavage or digestion within the patient. Any suitable linkage can beemployed which will be cleaved or digested following administration.

However, there is no intention on the part of the applicants to excludeother forms of administration.

In further embodiments of the invention, restoring nerve function in ananimal can comprise administering a therapeutic amount of a compound ofthe invention in combination with another neuroprotective agent,selected from, for example, growth factors and associated derivatives(insulin-like growth factor-I [IGF-I], insulin-like growth factor-II[IGF-II], transforming growth factor-β1, activin, growth hormone, nervegrowth factor, growth hormone binding protein, IGF-binding proteins[especially IGFBP-3], basic fibroblast growth factor, acidic fibroblastgrowth factor, the hst/Kfgk gene product, FGF-3, FGF-4, FGF-6,keratinocyte growth factor, androgen-induced growth factor. Additionalmembers of the FGF family include, for example, int-2, fibroblast growthfactor homologous factor-1 (FHF-1), FHF-2, FHF-3 and FHF-4, karatinocytegrowth factor 2, glial-activating factor, FGF-10 and FGF-16, ciliaryneurotrophic factor, brain derived growth factor, neurotrophin 3,neurotrophin 4, bone morphogenetic protein 2 [BMP-2], glial-cell linederived neurotrophic factor, activity-dependant neurotrophic factor,cytokine leukaemia inhibiting factor, oncostatin M, interleukin), α-,β-, γ-, or consensus interferon, and TNF-α. Other forms ofneuroprotective therapeutic agents include, for example, clomethiazole;kynurenic acid, Semax, tacrolimus,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol,andrenocorticotropin-(4-9) analogue [ORG 2766] and dizolcipine [MK-801],selegiline; glutamate antagonists such as, NPS1506, GV1505260, MK-801,GV150526; AMPA antagonists such as2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), LY303070and LY300164; anti-inflammatory agents directed against the addressingMAdCAM-1 and/or its integrin α4 receptors (α4β1 and α4β7), such asanti-MAdCAM-1 mAb MECA-367 (ATCC accession no. HB-9478).

The compound can be administered by a sustained-release system. Suitableexamples of sustained-release compositions include semi-permeablepolymer matrices in the form of shaped articles, e.g., films, ormicrocapsules. Sustained-release matrices include polylactides (U.S.Pat. No. 3,773,919; EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., 1983, Biopolymers: 22: 547-56),poly(2-hydroxyethyl methacrylate) (Langer et al., 1981, J. Biomed.Mater. Res.: 15: 267), ethylene vinyl acetate (Langer et al., 1981, J.Biomed. Mater. Res.: 15: 267), or poly-D-(−)-3-hydroxybutyric acid (EP133,988). Sustained-release compositions also include a liposomallyentrapped compound. Liposomes containing the compound are prepared bymethods known per se: DE 3,218,121, EP 52,322, EP 36,676, EP 88,046, EP143,949, EP 142,641, Japanese Pat. Appln. 83-118008, U.S. Pat. Nos.4,485,045 and 4,544,545, and EP 102,324. Ordinarily, the liposomes areof the small (from or about 200 to 800 Angstroms) unilamellar type inwhich the lipid content is greater than about 30 mol percentcholesterol, the selected proportion being adjusted for the mostefficacious therapy.

For parenteral administration, in one embodiment the compound isformulated generally by mixing each at the desired degree of purity, ina unit dosage injectable form (solution, suspension, or emulsion), witha pharmaceutically, or parenterally, acceptable carrier, i.e., one thatis non-toxic to recipients at the dosages and concentrations employedand is compatible with other ingredients of the formulation.

Generally, the formulations are prepared by contacting the compounduniformly and intimately with liquid carriers or finely divided solidcarriers or both. Then, if necessary, the product is shaped into thedesired formulation. Preferably the carrier is a parenteral carrier,more preferably a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, a buffered solution, and dextrose solution.Non-aqueous vehicles such as fixed oils and ethyl oleate are also usefulherein.

A carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; glycine; amino acids such as glutamic acid,aspartic acid, histidine, or arginine; monosaccharides, disaccharides,and other carbohydrates including cellulose or its derivatives, glucose,mannose, trehalose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; counter-ions such as sodium;non-ionic surfactants such as polysorbates, poloxamers, or polyethyleneglycol (PEG); and/or neutral salts, e.g., NaCl, KCl, MgCl₂, CaCl₂, etc.

A compound is typically formulated in such vehicles at a pH of from orabout 4.5 to 8. It will be understood that use of certain of theforegoing excipients, carriers, or stabilizers will result in theformation of salts of the compound. The final preparation can be astable liquid or lyophilized solid.

Formulations of the compound in pharmaceutical compositions can alsoinclude adjuvants. Typical adjuvants which can be incorporated intotablets, capsules, and the like are a binder such as acacia, cornstarch, or gelatin; an excipient such as microcrystalline cellulose; adisintegrating agent like corn starch or alginic acid; a lubricant suchas magnesium stearate; a sweetening agent such as sucrose or lactose; aflavouring agent such as peppermint, wintergreen, or cherry. When thedosage form is a capsule, in addition to the above materials, it canalso contain a liquid carrier such as a fatty oil. Other materials ofvarious types can be used as coatings or as modifiers of the physicalform of the dosage unit. A syrup or elixir can contain the activecompound, a sweetener such as sucrose, preservatives like propylparaben, a colouring agent, and a flavouring agent such as cherry.Sterile compositions for injection can be formulated according toconventional pharmaceutical practice. For example, dissolution orsuspension of the active compound in a vehicle such as water ornaturally occurring vegetable oil like sesame, peanut, or cottonseed oilor a synthetic fatty vehicle like ethyl oleate or the like may bedesired. Buffers, preservatives, antioxidants, and the like can beincorporated according to accepted pharmaceutical practice.

For injection, intraventricular administration, and other invasiveroutes of administration, the compounds used must be sterile. Sterilitycan be accomplished by any method known in the art, for examplefiltration through sterile filtration membranes (e.g., 0.2 micronmembranes). Therapeutic compositions generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper able to be pierced by a hypodermicinjection needle.

A pharmaceutical formulation ordinarily will be stored in unit ormulti-dose containers, for example, in sealed ampoules or vials, as anaqueous solution or as a lyophilized formulation for reconstitution. Asan example of a lyophilized formulation, 10 mL vials are filled with 5mL of sterile-filtered 1% (w/v) aqueous solution of compound, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized compound using bacteriostaticWater-for-Injection. It can be readily appreciated that other dosageforms and types of preparations can be used, and all are considered tobe part of this invention.

Preparation of the Compounds of this Invention

Starting materials and reagents used in preparing these compounds areeither available from commercial suppliers such as Aldrich ChemicalCompany (Milwaukee, Wis.), Bachem (Torrance, Calif.), Sigma (St. Louis,Mo.), Strem (Newburyport, Ma.) or are prepared by methods well known tothe person of ordinary skill in the art following procedures describedin such references as Fieser and Fieser's Reagents for OrganicSynthesis, vols 1-17, John Wiley and Sons, New York, N.Y., 1991; Rodd'sChemistry of Carbon Compounds, vols. 1-5 and supplements, ElsevierScience Publishers, 1989; Organic Reactions, vols. 1-40, John Wiley andSons, New York, N.Y., 1991; March J ; Advanced Organic Chemistry, 4^(th)ed. John Wiley and Sons, New York, N.Y., 1992; and Larock: ComprehensiveOrganic Transformations, VCH Publishers, 1989. In most instances, aminoacids and their esters or amides, and protected amino acids, are widelycommercially available; and the preparation of modified amino acids andtheir amides or esters are extensively described in the chemical andbiochemical literature and thus well-known to persons of ordinary skillin the art.

Starting materials, intermediates, and compounds of this invention canbe isolated and purified using conventional techniques, includingfiltration, distillation, crystallization, chromatography, and the like.They can be characterized using conventional methods, including physicalconstants and spectral data.

All patent and literature references cited throughout the specificationare expressly incorporated by reference in their entirety.

EXAMPLES

The present invention is further illustrated by the following examples.These examples are offered by way of illustration only and are notintended to limit the invention in any manner.

General Methods

Flash chromatography was performed using Scharlau 60 (40-60 μm mesh)silica gel. Analytical thin layer chromatography was carried out on 0.20mm pre-coated silica gel plates (ALUGRAM® SIL G/UV₂₅₄) and compoundsvisualized using UV fluorescence, or heating of plates dipped inpotassium permanganate in alkaline solution.

Melting points in degrees Celsius (° C.) were determined on anElectrothermal® melting point apparatus and are uncorrected.

Optical rotations were measured at 20° C. on a Perkin Elmer 341polarimeter using 10 cm path length cells and are given in units of 10⁻¹degcm²g⁻¹. Samples were prepared in the solvent indicated at theconcentration specified (measured in g/100 cm³). IR spectra wererecorded on a Perkin Elmer Spectrum One FT-IR spectrometer. The sampleswere prepared as thin films on sodium chloride discs or as solids inpotassium bromide discs. A broad signal indicated by br. The frequencies(ν) as absorption maxima are given in wavenumbers (cm⁻¹).

NMR spectra were recorded on a Bruker AVANCE DRX 400 (¹H, 400 MHz; ¹³C,100 MHz) or a Bruker AVANCE 300 (¹H, 300 MHz; ¹³C, 75 MHz) spectrometerat ambient temperatures. For ¹H NMR data chemical shifts are describedin parts per million downfield from SiMe₄ and are reported consecutivelyas position (δ_(H)), relative integral, multiplicity (s=singlet,d=doublet, t=triplet, dd=doublet of doublets, m=multiplet, br=broad),coupling constant (J/Hz) and assignment. For ¹³C NMR data, chemicalshifts are described in parts per million relative to CDCl₃ and arereported consecutively as position (δ_(C)), degree of hybridization asdetermined by DEPT experiments, and assignment. ¹H NMR spectra werereferenced internally using SiMe₄ (δ0.00) or CDCl₃ (δ 7.26). ¹³C NMRspectra were referenced internally using CDCl₃ (δ 77.0). When two setsof peaks arise in the NMR spectra due to different conformations aroundthe glycine-proline amide bond, the chemical shift for the minor cisconformer is marked with an asterisk (*).

Accurate mass measurements were recorded on a VG-70SE mass spectrometer.

Hexane and dichloromethane were distilled prior to use. Methanol wasdried using magnesium turnings and iodine, and distilled under nitrogen.Triethylamine was dried over calcium hydride and distilled undernitrogen.

Analytical HPLC was performed on a Waters 600 system using an AlltechEconosphere C18 RP column (150 mm×4.6 mm). A 5 minute flush with water(containing 0.05% trifluoroacetic acid) followed by gradient elution ofwater (containing 0.05% trifluoroacetic acid) to acetonitrile over 25mins was used at a flow rate of 1 ml min⁻¹. For semi-preparativepurification an Alltech Econosil C18 RP column (250 mm×22 mm) was usedunder isocratic conditions, typically 80% water (containing 0.05%trifluoroacetic acid):20% acetonitrile at a flow rate of 13 ml min⁻¹.

Example 1 Synthesis of(2S,3′S,8′S)-2-{[(3′-Amino-1′-aza-2′-oxobicyclo[6.3.0]undecyl)-8′-carbonyl]amino}-1,5-pentanedioicacid trifluoroacetate salt

N-tert-Butoxy-(S)-allylglycine 2

A solution of allylglycine 1 (0.1 g, 0.87 mmol) and sodium hydrogencarbonate (0.08 g, 0.96 mmol) in water/dioxane (1:1, v/v, 4 cm³) wascooled to 0° C., di-tert-butyl dicarbonate (0.07 g, 0.32 mmol) was addedand the solution stirred for 2 h at 0° C. Di-tert-butyl dicarbonate(0.07 g, 0.32 mmol) was then added and the solution stirred for afurther 2 h at 0° C., after which time further di-tert-butyl dicarbonate(0.1 g, 0.46 mmol) was added and the solution allowed to warm to roomtemperature and stirred overnight. Dioxane was removed under reducedpressure at 40° C. and the resultant residue was dissolved in water,washed with ether and the aqueous layer acidified with 2M aqueoushydrochloric acid and extracted with ethyl acetate. The combined organiclayers were dried (MgSO₄), filtered and the solvent removed int vacuo togive crude carbamate 2 (0.14 g, ca. 72%) as a colourless oil. Thismaterial was used as such for the subsequent step. Carbamate 2 was shownto be a 73:27 mixture of rotamers: δ_(H) (400 MHz; CDCl₃; Me₄Si) 1.45[9H, s, C(CH₃)₃], 2.51-2.63 [2H, m, CH₂(allyl)], 4.19* (0.27H, br s,2-H), 4.41 (0.73H, d, J 6.1, 2-H), 5.13-5.20 (2.73H, m, ═CH₂, N—H),5.70-5.80 [1H, m, C(H)═CH₂], 6.26 (0.27H, br s, N—H*) and 9.16 (1H, brs, OH); δ_(C) (100 MHz; CDCl₃) 28.2 [CH₃, C(CH₃)₃], 36.3 [CH₂,CH₂(allyl)], 52.7 (CH, 2-C), 54.2* (CH, 2-C), 80.2 [quat, C(CH₃)₃],81.6* [quat., C(CH₃)₃], 119.2 (CH₂, ═CH₂), 132.1 [CH, C(H)═CH₂], 155.4(quat., NCO₂), 155.4* (quat., NCO₂), and 176.1 (quat., 1-CO).

N-tert-Butyloxy-(S)-allylglycl-(S)-allylproline methyl ester 4

N,N′-Dicyclohexylcarbodiimide (0.1 g, 0.49 mmol) was added to a stirredsolution of (S)-allylproline methyl ester 3 (0.1 g, 0.49 mmol),N-tert-butoxy-(S)-allylglycine 2 (0.12 g, 0.54 mmol),N-hydroxybenzotriazole (0.065 g, 0.486 mmol) and triethylamine (0.07cm³, 0.486 mmol) in dichloromethane (10 cm³) at 0° C. The mixture wasstirred overnight at room temperature, refrigerated for 2 h and filteredthrough Celite™ to remove dicyclohexyl urea. The filtrate was washedwith saturated aqueous sodium hydrogen carbonate, 2 M aqueoushydrochloric acid, dried (Na₂SO₄), filtered and the solvent removed toyield an oil which was purified by chromatography (SiO₂, 4:1, 3:1,hexane-ethyl acetate) to afford diene 4 (0.041 g, ca. 23%) as acolourless oil. Diene 4 existed exclusively as the trans GlyC(O)—NProconformer: δ_(H) (400 MHz; CDCl₃; Me₄Si) 1.43 [9H, s, C(CH₃)₃],1.94-2.15 (4H, m, Proβ-H₂, Proγ-H₂), 2.35 [1H, p, J 7.1,allyl-H_(A)H_(B)(Pro)], 2.50 [1H, p, J 7.2, allylH_(A)H_(B)(Pro)], 2.62[1H, dd, J 14.0 and 8.4, allyl-H_(A)H_(B)(Gly)], 3.18 [1H, dd, J 14.1and 6.5, allyl-H _(A)H_(B)(Gly)], 3.65-3.80 (5H, m, OCH₃ and Proδ-H₂),4.46 (1H, dd, J 15.4 and 6.7, Glyα-H), 5.08-5.25 (5H, m, 2×═CH₂ and N—H)and 5.63-5.86 (2H, m, 2×C(H)═CH₂); δ_(C) (100 MHz; CDCl₃) 23.6 (CH₂,Proγ-C), 28.2 [CH₃, C(CH₃)₃], 35.0 (CH₂, Proδ-C), 36.3 [CH₂,CH₂(allyl)Gly], 37.7 [CH₂, CH₂(allyl)Pro], 48.5 (CH₂, Proδ-C), 51.6 (CH,Glyα-C), 52.2 (CH₃, OCH₃), 68.5 (quat., Proα-C), 79.6 [quat., C(CH₃)₃],118.5 (CH₂, ═CH₂), 119.1 (CH₂, ═CH₂), 132.7 [CH, C(H)═CH₂], 133.0 [CH,C(H)═CH₂], 155.4 (quat., NCO₂), 170.2 (quat., Gly-CO) and 173.7 (quat.,Pro-CO).

(3S,8S)-1-Aza-3-(tert-butyloxycarbonlylamino)-8-methoxycarbonyl-2-oxobicyclo[6.3.0]undec-5-ene5

A solution of freshly sublimed potassium tert-butoxide (0.0018 g, 0.0157mmol) in dry tetrahydrofuran (1 cm³) was added to a stirred suspensionof 1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydro-imidazoliumtetrafluoroborate (0.07 g, 0.0177 mmol) in dry tetrahydrofuran (2 cm³)under an atmosphere of nitrogen. Dry tetrahydrofuran (1 cm³) was used torinse the remaining potassium tert-butoxide from the reaction flask. Theresultant suspension was stirred for 2 min then a solution ofbis(tricyclohexylphosphine)benzylidineruthenium dichloride (Grubbs'scatalyst) (0.010 g, 0.011 mmol) in dry benzene (10 cm³) was added andthe purple solution heated at 80° C. for 35 min. The dark brown solutionwas cooled to room temperature and a solution of diene 4 (0.041 g, 0.112mmol) in dry benzene (40 cm³) added and the mixture heated at 45° C. for48 h. The brown solution was cooled to room temperature, dimethylsulphoxide (0.043 g, 0.56 mmol) was added and the mixture stirredovernight. The solvent was removed in vacuo and the residue purified bychromatography (SiO₂, 2:1, 1:1, hexane-ethyl acetate) to give alkene 5(0.022 g, 58%) as a greenish oil. Alkene 5 existed exclusively as thetrans C(O)—NPro conformer: [α]_(D) −86.3 (c 0.183 in CH₂Cl₂) [lit.²,−87.4 (c 0.35 in CHCl₃]; δ_(H) (400 MHz; CDCl₃; Me₄Si) 1.44 [9H, s,C(CH₃)₃], 1.66-1.86 (2H, m, 10-H₂), 1.96-2.11 (2H, m, 9-H_(A)H_(B) and4-H_(A)H_(B)), 2.46 (1H, dd, J 12.3 and 6.1, 9-H_(A)H_(B)), 2.66 (1H,dd, J 14.8 and 8.2, 7-H_(A)H_(B)), 2.80-2.90 (1H, m, 4-H_(A)H_(B)), 3.04(1H, dd, J 15.1 and 8.6, 7-H_(A)H_(B)), 3.40 (1H, ddd, J 11.6, 11.1 and7.4, 11-H_(A)H_(B)), 3.76-3.83 (1H, m, 11-H_(A)H_(B)), 3.78 (3H, s,OCH₃), 4.97-5.04 (1H, m, 3-H), 5.52-5.63 (2H, m, 6-H and N—H) and5.74-5.79 (1H, m, 5-H); δ_(C) (100 MHz; CDCl₃) 20.6 (CH₂, 10-C), 28.2[CH₃, C(CH₃)₃], 35.0 (CH₂, 7-C), 35.4 (CH₂, 4-C), 38.4 (CH₂, 9-C), 48.4(CH₂, 11-C), 50.8 (CH, 3-C), 53.0 (CH₃, OCH₃), 69.7 (quat., 8-C), 79.4[quat., C(CH₃)₃], 122.8 (CH, 5-C), 132.2 (CH, 6-C), 154.8 (quat, NCO₂),171.5 (quat., 2-C) and 173.7 (quat., 8-CO).

(2S,3′S,8S′)-Di-tert-butyl2-{[(1′-aza-3′-(tert-butyloxycarbonylamino)-2′-oxobicyclo[6.3.0]undec-5′-ene)-8′-carbonyl]amino}-1,5-pentadioate7

To a solution of alkene 5 (0.022 g, 0.065) in dioxane (0.7 cm³) wasadded 1 M aqueous sodium hydroxide (0.32 cm³, 0.32 mmol) and the opaquesolution stirred at room temperature for 17 h. Water was added and themixture washed with dichloromethane. The aqueous layer was acidifiedwith citric acid and the product extracted with dichloromethane. Theorganic layers were pooled, dried (MgSO₄) and the solvent removed toafford an oil (0.020 g). To a solution of this oil in dichloromethane (4cm³) was added L-glutamic acid di-tert-butyl ester p-toluenesulphonate 6(0.025 g, 0.085 mmol) was added and the solution cooled to 0° C.Triethylamine (0.017 g, 0.17 mmol) andbis(2-oxo-3-oxazolidinyl)phosphinic chloride (0.021 g, 0.085 mmol) wereadded and the solution stirred for 20 h. The reaction mixture was washedwith 2M aqueous hydrochloric acid, saturated aqueous sodium hydrogencarbonate, dried (Na₂SO₄), filtered and the solvent removed to yield anoil (0.041 g) which was purified by chromatography (SiO₂, 1:1, 2:3,hexane-ethyl acetate) to give amide 7 (0.025 g, 69% in 2 steps) as acolorless oil. Amide 7 existed exclusively as the cis C(O)—NProconformer: [α]_(D) −49.6 (c 0.25 in CH₂Cl₂); δ_(H) (400 MHz; CDCl₃;Me₄Si) 1.40-1.44 [27H, m, 3×C(CH₃)₃], 1.55-1.66 (1H, m), 1.78 (1H, p, J6.4), 1.89 (1H, td, J 13.2 and 6.1), 2.05-2.39 (5H, m), 2.65-2.75 (2H,m, 4′-H₂), 2.80 (1H, dd, J 15.0 and 7.9, 7′-H_(A)H_(B)), 2.92 (1H, dd, J14.9 and 8.9, 7′-H_(A)H_(B)), 3.55 (1H, ddd, J 11.8, 11.8 and 7.0,11′-H_(A)H_(B)), 3.65-3.70 (1H, m, 11′-H_(A)H_(B)), 4.40-4.46 (1H, m,3′-H), 4.88-4.97 (2H, m, 6′-H and 2-H), 5.67-5.79 (2H, m, 5′-H and N—H)and 8.36 (1H, d, J 8.4, N—H); Bc (100 MHz; CDCl₃) 20.4 (CH₂, 10′-C),25.4 (CH₂, 3-C), 27.9 [CH₃, C(CH₃)₃], 28.0 [CH₃, C(CH₃)₃], 28.2 [CH₃,C(CH₃)₃], 31.5 (CH₂, 4-C), 32.2 (CH₂, 4′-C), 34.6 (CH_(2,7)′-C), 37.4(CH₂, 9′-C), 49.3 (CH₂, 11′-C), 52.1 (CH, 3′-C), 52.8 (2-C), 71.8(quat., 8′-C), 80.4 [quat., C(CH₃)₃], 81.1 [quat., C(CH₃)₃], 125.0 (CH,5′-C), 130.2 (CH, 6′-C), 156.3 (quat., NCO₂), 170.5 (quat., 2′-C), 171.9(quat., 8′-CO), 171.2 (quat., 1-C) and 173.5 (quat., 5-C); m/z (EI+)565.3365 (M⁺. C₂₉H₄₇N₃O₈ requires 565.3363).

(2S,3′S,8′S)2-{[(1′-Aza-3′-amino-2′-oxobicyclo[6.3.0]undecyl)-8′-carbonyl]amino}-1,5-pentandioicacid trifluoroacetate salt 8

PtO₂ (0.001 g, 0.004 mmol) was added to a stirred solution of amide 7(0.025 g, 0.044 mmol) in tetrahydrofuran (4 cm³) under a nitrogenatmosphere. The mixture was hydrogenated (1 atm. of hydrogen) for 17 h,filtered through Celite™, and the solvent removed in vacuo. The residuewas dissolved in dichloromethane (3 cm³), trifluoroacetic acid (1 cm³)added and the solution stirred for 4 h at room temperature. Removal ofthe volatiles in vacuo and analysis by HPLC and NMR showed the reactionto be incomplete. The residue was therefore redissolved indichloromethane-trifluoroacetic acid (3:1, 4 cm³) and the solutionstirred at room temperature for 2.5 h. The volatiles were removed invacuo, and the residue purified by RP HPLC [20% acetonitrile:80% water(containing 0.05% trifluoroacetic acid)] to give an oil which wastrituated from ether/toluene to give 8 (0.0167 g, 81%, in 2 steps) as ahygroscopic white solid. Macrocycle 8 existed exclusively as the cisC(O)—NPro conformer: mp 75-85° C.; [α]_(D) −4.4 (c 0.16 in MeOH); δ_(H)(400 MHz; D₂O) 1.30-1.39 (1H, m, 5′-H_(A)H_(B)), 1.57-1.65 (1H, m,6′-H_(A)H_(B)), 1.79-1.91 (4H, m, 5′-H_(A)H_(B), 6′-H_(A)H_(B),4′-H_(A)H_(B) and 10′-H_(A)H_(B)), 1.98-2.13 (4H, m, 4′-H_(A)H_(B),10′-H_(A)H_(B), 7′-H_(A)H_(B) and 3-H_(A)H_(B)), 2.23-2.37 (4H, m,9′-H₂, 3-H_(A)H_(B) and 7′-H_(A)H_(B)), 2.53 (2H, t, J 7.5, 4-H₂), 3.68(1H, dt, J 14.2 and 7.4, 11′-H_(A)H_(B)), 3.79 (1H, dt, J 14.0 and 7.5,11′-H_(A)H_(B)), 4.20 (1H, dd, J 11.2 and 6.3, 3-H) and 4.53 (1H, dd, J10.2 and 4.9, 2-H); δ_(C) (100 MHz; D₂O) 20.4 (CH₂, 10′-C), 21.2 (CH₂,6′C), 22.1 (CH₂, 5′-C), 24.8 (CH₂, 3-C), 30.3 (CH₂, 4-C), 31.1 (CH₂,4′-C), 36.4 (CH₂, 7′-C), 40.9 (CH₂, 9′-C), 49.1 (CH₂, 11′-C), 51.2 (CH,3′-C), 52.4 (CH, 2-C), 70.9 (quat., 8′-C), 116.1 (quat., q, J 291, CF₃),162.7 (quat., q, J 36.0, CF₃CO₂H), 169.4 (quat., 2′-C), 174.6 (quat.,8′-CO), 175.3 (quat., 1-C) and 177.0 (quat., 5-C); m/z (FAB+) 356.1830[MH(free base)⁺. C₁₆H₁₆N₃O₆ requires 356.1822].

Example 2 Synthesis of(2S,9′S,12′S)-2-{[(1′,4′-Diaza-2′-oxobicyclo[7.3.0]dodecyl)-12′-carbonyl]amino}-1,5-pentanedioicacid trifluoroacetate salt

(S)-tert-Butyl pyroglutamate 10

Perchloric acid (70% aqueous, 0.521 cm³, 6.05 mmol) was added dropwiseto a stirred suspension of (S)-pyroglutamic acid 9 (1 g, 7.75 mmol) intert-butyl acetate (12 cm³, 85.5 mmol). The mixture was stirred for 24 hthen sodium carbonate (0.80 g, 7.60 mmol) was added cautiously, etherwas added and the organic phase washed with saturated sodium hydrogencarbonate solution and brine. The aqueous phase was extracted with ethylacetate, dried (MgSO₄), filtered and the solvent removed to yield an oilto which was added carbon tetrachloride and the solvent evaporated invacuo to remove traces of acetic acid to give ester 10 (0.81 g, 56%) asa white solid: δ_(H) (300 MHz; CDCl₃; Me₄Si) 1.48 [9H, s, C(CH₃)],2.33-2.45 (4H, m, 3-H₂, 4-H₂), 4.12-4.16 (1H, m, 5-H) and 6.16 (1H, brs, N—H); δ_(C) (75 MHz; CDCl₃) 24.7 (CH₂, 4-C), 27.8 [CH₃, C(CH₃)], 29.2(CH₂, 3-C), 55.9 (CH, 5-C), 82.3 [quat., C(CH₃)], 170.9 (quat., 5-CO)and 177.7 (quat., 2-C).

(S)-tert-Butyl N-tert-butoxycarbonylpyroglutamate 11

To a solution of ester 10 (0.85 g, 4.58 mmol) and dimethylaminopyridine(0.056 g, 0.458 mmol) in acetonitrile (40 cm³) was added di-tert-butyldicarbonate (1.12 g, 5.50 mmol). The resultant solution was stirred atroom temperature for 19 h, concentrated in vacuo and purified bychromatography (SiO₂, 4:1, 3:1 hexane-ethyl acetate; gradient elution)to give carbamate 11 (1.24 g, 95%) as a white solid: mp 53-55° C. (lit.54-56° C.); δ_(H) (300 MHz; CDCl₃; Me₄Si) 1.48 [9H, s, C(CH₃)₃], 1.50[9H, s, C(CH₃)₃], 1.93-2.03 (1H, m), 2.16-2.35 (1H, m), 2.40-2.66 (2H,m) and 4.47 (1H, dd, J 9.4 2.7, 5-H); δ_(C) (75 MH; CDCl₃) 21.6 (CH₂,4-C), 27.8 [CH₃, C(CH₃)₃], 31.0 (CH₂, 3-C), 59.5 (CH, 5-C), 82.2 [quat.,C(CH₃)], 83.2 [quat., C(CH₃)₃], 149.2 (quat., NCO₂), 170.3 (quat., 5-CO)and 173.4 (quat., 2-C).

N-tert-Butoxycarbonyl-5-allyl-L-proline tert-butyl ester 13

To a stirred solution of carbamate 11 (1.14 g, 3.98 mmol) in drytetrahydrofuran (40 cm³) was added a 1 mol L⁻¹ tetrahydrofuran solutionof lithium triethylborohydride (4.78 cm³, 4.78 mmol) at −78° C. under atatmosphere of nitrogen. The solution was stirred for 1 h, saturatedaqueous sodium hydrogen carbonate (10 cm³) added and the cooling bathremoved. The temperature was allowed to reach 0° C. then 30% aqueoushydrogen peroxide (30 drops) was added and stirring was continued at 0°C. for 30 min. The aqueous layer was extracted with ether and thecombined organic extracts were washed with brine, dried (MgSO₄),filtered and the solvent removed to give alcohol 12 (1.17 g) whichcontained ˜8% of 11 as determined by ¹H NMR. The crude alcohol 12 wasdissolved in dry dichloromethane (20 cm³) and added dropwise to astirred solution of trimethylsilyl triflate (1.44 cm³, 7.96 mmol) andallyltributylstannane (2.46 cm³, 7.96 mmol) in dry dichloromethane (20cm³) at −78° C. under nitrogen. The solution was stirred at −78° C. for2 h, saturated aqueous sodium hydrogen carbonate (15 cm³) was added andthe reaction warmed to room temperature. The aqueous layer was extractedwith dichloromethane and the pooled organic extracts dried (MgSO₄) andfiltered. Removal of the solvent in vacuo gave an oil (3.855 g) whichwas purified by chromatography (SiO₂, 8:1, 7:1, hexane-ethyl acetate;gradient elution) to afford an oil (1.047 g) contaminated withtributylstannane residues. Further chromatography (SiO₂, 12:1, 10:1,8:1, hexanes-ethyl acetate; gradient elution) gave alkene 13 (0.872 g,70%, in 2 steps) as a colourless oil. Alkene 13 was shown to be a 57:43mixture of C(2)/C(5) cis:trans isomers: δ_(H) (300 MHz; CDCl₃; Me₄Si)1.41-1.45 [18H, m, 2×C(CH₃)₃], 1.67-2.24 [5H, m, Proβ-H₂, Proγ-H₂ andCH_(A)CH_(B)(allyl)], 2.39-2.71 [1H, m, CH_(A)CH_(B)(allyl)], 3.79-4.18(2H, m, Proα-H and Proδ-H), 4.99-5.08 (2H, m, ═CH₂) and 5.67-5.85 [1H,m, C(H)═CH₂]; δ_(C) (75 MHz; CDCl₃) 26.6 (CH₂, Proγ-C), 27.4 (CH₂,Proγ-C), 27.8 (CH₂, Proγ-C), 27.86 [CH₃, C(CH₃)₃], 27.92 [CH₃, C(CH₃)₃],28.25 [CH₃, C(CH₃)₃], 28.31 [CH₃, C(CH₃)₃], 28.4 (CH₂, Proβ-C), 29.4(CH₂, Proβ-C), 30.2 (CH₂, Proβ-C), 38.1 [CH₂, CH₂(allyl)], 38.5 [CH₂,CH₂(allyl)], 38.98 [CH₂, CH₂(allyl)], 39.1 [CH₂, CH₂(allyl)], 57.4 (CH,Proδ-C), 58.0 (CH, Proδ-C), 60.6 (CH, Proα-C), 60.9 (CH, Proα-C), 79.5[quat., C(CH₃)₃], 79.6 [quat., C(CH₃)₃], 80.7 [quat., C(CH₃)₃], 80.74[quat, C(CH₃)₃], 116.9 (CH₂, ═CH₂), 116.99 (CH₂, ═CH₂), 117.0 (CH₂,═CH₂), 135.12 (CH, C(H)═CH₂), 135.2 (CH, C(H)═CH₂), 135.5 (CH,C(H)═CH₂), 153.7 (quat., NCO₂), 153.8 (quat., NCO₂), 154.3 (quat.,NCO₂), 172.1 (quat., Proα-CO), 172.2 (quat., Proα-CO) and 172.3 (quat.,Proα-CO); m/z (EI+) 312.2171 (M⁺. C₁₇H₃₀NO₄ requires 312.2175).

(1S,2S)-5-Allylproline tert-butyl ester (trans) 14 and (1S,2R)5-allylproline tert-butyl ester (cis) 15

To alkene 13 (0.24 g, 0.77 mmol) was added a 4 mol L⁻¹ solution ofhydrogen chloride in dioxane (4 cm³) at 0° C. The solution was stirredfor 1 h at 0° C. then room temperature for 40 min at which time reactionwas complete (by tlc). The mixture was cooled to 0° C., neutralized witha saturated solution of aqueous sodium hydrogen carbonate and theproduct extracted with dichloromethane. Removal of the solvent in vacuoyielded an oil (0.142 g) which was purified by chromatography (SiO₂,2:1, 5:4, 1:1, hexanes-ethyl acetate) to give (i) amine 14 (trans)(0.051 g, 31%) as a colourless oil. This compound showed no NOE betweenthe Proα-H atom at δ3.70 and the Proδ-H atom at δ3.26: [α]_(D) −31 (c0.47 in CH₂Cl₂); δ_(H) (300 MHz; CDCl₃; Me₄Si) 1.35-1.41 (1H, m,Proγ-H_(A)H_(B)), 1.43 [9H, m, C(CH₃)₃], 1.72-1.90 (2H, Proγ-H_(A)H_(B)and ProβH_(A)H_(B)), 2.11-2.20 [3H, m, ProβH_(A)H_(B) and CH₂(allyl)],2.47 (1H, s, N—H), 3.26 (1H, p, J 6.5, Proδ-H), 3.70 (1H, dd, J 8.5 and5.9, Proα-H), 4.98-5.10 (2H, m, ═CH₂) and 5.73-5.84 [1H, m, C(H)═CH₂];δ_(C) (75 MHz; CDCl₃) 27.9 [CH₃, C(CH₃)₃], 29.5 (CH₂, Proβ-C), 30.7(CH₂, Proγ-C), 40.8 [CH₂, CH₂(allyl)], 57.6 (CH, Proδ-C), 59.7 (CH,Proα-C), 80.7 [quat., C(CH₃)₃], 116.1 (CH₂, ═CH₂), 136.0 [CH, C(H)═CH₂],and 174.9 (quat., Proα-CO); (ii) amine 15 (cis) (0.066 g, 41%) as acolourless oil. This compound showed an NOE between the Proα-H atom atδ3.57-3.62 and the Proδ-H atom at δ3.03-3.12: [α]_(D) −22.1 (c 0.66 inCH₂Cl₂); δ_(H) (300 MHz; CDCl₃; Me₄Si) 1.24-1.30 (1H, m,Proγ-H_(A)H_(B)), 1.42 [9H, m, C(CH₃)₃], 1.78-1.87 (2H, Proγ-H_(A)H_(B)and Proβ-H_(A)H_(B)), 1.98-2.07 (1H, m, Proβ-H_(A)H_(B)), 2.18-2.31 [3H,m, CH₂(allyl) and N—H), 3.03-3.12 (1H, m, Proδ-H), 3.57-3.62 (1H, m,Proα-H), 4.99-5.1 (2H, m, ═CH₂) and 5.72-5.86 [1H, m, C(H)═CH₂]; δ_(C)(75 MHz; CDCl₃) 27.9 [CH₃, C(CH₃)₃], 30.4 (CH₂, Proβ-C), 31.1 (CH₂,Proγ-C), 39.9 [CH₂, CH₂(allyl)], 59.1 (CH, Proδ-C), 60.5 (CH, Proα-C),80.8 [quat., C(CH₃)], 116.5 (CH₂, ═CH₂), 135.5 [CH, C(H)═CH₂], and 174.3(quat., Proα-CO); m/z (CI+) 212.1647 (MH⁺. C₁₂H₂₂NO₂ requires 212.1651).

(1S,2S)-N-Allyl-N-benzyloxycarbonylglycine-5-allylproline tert-butylester (trans) 18 and(1S,2R)N-allyl-N-benzyloxycarbonylglycine-5-allylproline tert-butylester (cis) 17

To alkene 13 (0.87 g, 0.77 mmol) was added a 4M solution of hydrogenchloride in dioxane (4 cm³) at 0° C. The solution was stirred for 1 h at0° C. then room temperature for 35 min after which time reaction wascomplete (by tlc). The mixture was cooled to 0° C., neutralized with asaturated solution of aqueous sodium hydrogen carbonate and the productextracted with dichloromethane. Removal of the solvent in vacuo yieldedan oil (0.568 g) that was dissolved in dichloromethane (30 cm³). To thiswas added triethylamine (0.54 cm³, 4.0 mmol),N-allyl-N-benzyloxycarbonylglycine 16 (1.0 g, 4.0 mmol) and1-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride) (EDCI) (0.77g, 4.0 mmol) at 0° C. and the solution stirred for 16 h. The mixture waswashed with saturated aqueous sodium hydrogen carbonate, 2 M aqueoushydrochloric acid, dried (MgSO₄), filtered and the solvent removed toyield an oil (1.7 g) which was purified by chromatography (SiO₂, 5:1,4:1, 3:1, 2:1, hexane-ethyl acetate) to afford (i) diene 18 (trans)(0.29 g, 24%) as a colourless oil. Diene 18 was shown to be a 1:1trans:cis mixture of GlyC(O)—NPro conformers. In addition, restrictedrotation about the GlyN—CO carbamate bond was also observed resulting ina 1:1 mixture of conformers: [α]_(D) −56.1 (c 0.77 in CH₂Cl₂); δ_(H)(400 MHz; CDCl₃; Me₄Si) 1.43 [9H, s, C(CH₃)₃], 1.46 [9H, s, C(CH₃)₃],1.48 [9H, s, C(CH₃)₃], 1.74-2.71 [6H, m, Proβ-H₂, Proγ-H₂ andCH₂(allyl)], 3.54 (0.5H, d, J 16.5, Glyα-H_(A)H_(B)), 3.58 (0.5H, d, J16.5, Glyα-H_(A)H_(B)), 3.84-4.40 [5H, m, Glyα-H CH₂(allyl), Proα-H andProδ-H], 4.98-5.25 (6H, m, OCH₂Ph and 2x ═CH₂), 5.56-5.88 [2H, m,2×C(H)═CH₂] and 7.30-7.37 (5H, m, Ph); δ_(C) (100 MHz; CDCl₃) 25.8 (CH₂,Proβ-C or Proγ-C), 25.9 (CH₂, Proβ-C or Proγ-C), 26.16 (CH₂, Proβ-C orProγ-C), 26.2 (CH₂, Proβ-C or Proγ-C), 27.8 [CH₃, C(CH₃)₃], 28.3 (CH₂,Proβ-C or Proγ-C), 29.3 (CH₂, Proβ-C or Proγ-C), 29.4 (CH₂, Proβ-C orProγ-C), 36.9 [CH₂, CH₂(allyl)], 37.0 [CH₂, CH₂(allyl)], 39.0 [CH₂,CH₂(allyl)], 39.3 [CH₂, CH₂(allyl)], 47.4 (CH₂, Glyα-C), 47.6 (CH₂,Glyα-C), 47.8 (CH₂, Glyα-C), 48.1 (CH₂, Glyα-C), 50.14 [CH₂,NCH₂(allyl)], 50.18 [CH₂, NCH₂(allyl)], 50.7 [CH₂, NCH₂(allyl)], 50.8[CH₂, NCH₂(allyl)], 56.9 (CH, Proδ-C), 57.1 (CH, Proδ-C), 58.2 (CH,Proδ-C), 58.3, (CH, Proδ-C), 60.0 (CH, Proα-C), 60.1 (CH, Proα-C), 60.2(CH, Proα-C), 60.3 (CH, Proα-C), 67.3 (CH₂, OCH₂Ph), 67.4 (CH₂, OCH₂Ph),81.1 [quat., C(CH₃)₃], 82.3 [quat., C(CH₃)₃], 116.9 (CH₂, ═CH₂), 117.1(CH₂, ═CH₂), 117.2 (CH₂, ═CH₂), 117.6 (CH₂, ═CH₂), 117.7 (CH₂, ═CH₂),118.1 (CH₂, ═CH₂), 118.2, (CH₂, ═CH₂), 127.6 (CH, Ph), 127.8 (CH, Ph),127.9 (CH, Ph), 128.2 (CH, Ph), 128.3 (CH, Ph), 133.3 [CH, C(H)═CH₂],133.4 [CH, C(H)═CH₂], 133.6 [CH, C(H)═CH₂], 133.8 [CH, C(H)═CH₂], 134.9[CH, C(H)═CH₂], 135.0 [CH, C(H)═CH₂], 136.5 (quat., Ph), 156.0 (quat.,NCO₂), 156.37 (quat., NCO₂), 156.42, (quat., NCO₂), 167.1 (quat.,Gly-CO), 167.3 (quat., Gly-CO), 167.4 (quat., Gly-CO), 167.6 (quat.,Gly-CO) and 170.9 (quat., Proα-CO), 171.0 (quat., Proα-CO), 171.1(quat., Proα-CO); m/z (EI+) 442.2455 (M⁺. C₂₅H₃₄N₂O₅ requires 442.2468);(ii) a mixture of 17 and 18 (0.044 g, 4%); (iii) diene 17 (cis) (0.424g, 36%) as a colourless oil. Diene 17 was shown to be a 1:1 trans:cismixture of GlyC(O)—NPro conformers. In addition, restricted rotationabout the GlyN—CO carbamate bond was also observed resulting in a 1:1mixture of conformers: [α]_(D) −39.2 (c 0.57 in CH₂Cl₂); δ_(H) (400 MHz;CDCl₃; Me₄Si) 1.43 [9H, s, C(CH₃)₃], 1.45 [9H, s, C(CH₃)₃], 1.47 [9H, s,C(CH₃)₃], 1.49 [9H, s, C(CH₃)₃], 1.69-2.49 [5.75H, m, Proβ-H₂, Proγ-H₂and CH₂(allyl)], 2.78-2.84 [0.25H, m, CH₂(allyl)], 3.61-4.41 [6H, m,Glyα-H₂, CH₂(allyl), Proα-H and Proδ-H), 5.02-5.12 (6H, m, OCH₂Ph and2×═CH₂), 5.67-5.87 [2H, m, 2x C(H)CH₂] and 7.29-7.34 (5H, m, Ph); δ_(C)(100 MHz; CDCl₃) 26.5 (CH₂, Proβ-C), 27.7 [CH₃, C(CH₃)₃], 27.8 [CH₃,C(CH₃)₃], 27.9 [CH₃, C(CH₃)₃], 29.4 (CH₂, Proβ-C or Proγ-C), 29.6 (CH₂,Proβ-C or Proγ-C), 29.7 (CH₂, Proβ-C or Proγ-C), 37.65 [CH₂,CH₂(allyl)], 37.7 [CH₂, CH₂(allyl)], 38.9 [CH₂, CH₂(allyl)], 47.2 (CH₂,Glyα-C), 47.6 (CH₂, Glyα-C), 47.7 (CH₂, Glyα-C), 48.0 (CH₂, Glyα-C),50.2 [CH₂, NCH₂(allyl)], 50.3 [CH₂, NCH₂(allyl)], 50.8 [CH₂,NCH₂(allyl)], 50.9 [CH₂, NCH₂(allyl)], 57.8 (CH, Proδ-C), 58.5 (CH,Proδ-O), 60.2, 60.4 (CH, Proα-C), 60.5 (CH, Proα-C), 67.3 (CH₂, OCH₂Ph),67.4 (CH₂, OCH₂Ph), 81.0 [quat., C(CH₃)₃], 82.2 [quat., C(CH₃)₃], 116.9(CH₂, ═CH₂), 117.0 (CH₂, ═CH₂), 117.5 (CH₂, ═CH₂), 118.0 (CH₂, ═CH₂),127.6 (CH, Ph), 127.8 (CH, Ph), 127.9 (CH, Ph), 128.0 (CH, Ph), 128.3(CH, Ph), 133.3 [CH, C(H)═CH₂], 133.4 [CH, C(H)═CH₂], 133.5 [CH,C(H)═CH₂], 133.6 [CH, C(H)═CH₂], 134.1 [CH, C(H)═CH₂], 134.3 [CH,C(H)═CH₂], 134.8 [CH, C(H)═CH₂], 134.9 [CH, C(H)═CH₂], 136.5 (quat.,Ph), 155.8 (quat., NCO₂), 155.9 (quat., NCO₂), 156.38 (quat., NCO₂),156.44 (quat., NCO₂), 166.95 (quat., Gly-CO), 167.01 (quat., Gly-CO),167.4 (quat., Gly-CO), 167.5 (quat., Gly-CO), 171.0 (quat., Proα-CO),172.2 (quat., Proα-CO) and 171.3 (quat., Proα-CO); m/z (EI+) 442.2462(M⁺. C₂₅H₃₄N₂O₅ requires 442.2468).

(6Z,9S,12S)N-Benzyloxycarbonyl-1,4-diaza-12-tert-butoxycarbonyl-2-oxobicyclo[7.3.0]dodec-6-ene19

To a degassed solution of diene 17 (0.10 g, 0.23 mmol) in drydichloromethane (56 cm³) was addedbis(tricyclohexylphosphine)benzylidineruthenium dichloride (Grubbs'scatalyst) (0.018 g, 0.023 mmol) under an atmosphere of nitrogen and theresultant purple solution heated at reflux for 24 h. Furtherbis(tricyclohexylphosphine)benzylidineruthenium dichloride (0.018 g,0.023 mmol) was added and refluxing continued for 24 h, then thereaction mixture was cooled to room temperature and dimethyl sulphoxide(0.160 cm³, 2.3 mmol) was added and the orange/brown solution stirredfor 24 h. The solvent was removed in vacuo and the residue purified bychromatography (SiO₂, 3:1, 2:1, 1:1, hexanes-ethyl acetate) to give analmost colourless oil which was further purified by chromatography (C₁₈RP silica, 100:0, 9:1, 9:3, 1:1, 1:9, water-acetonitrile) to give alkene19 [0.039 g, 42% (60% based on recovered starting material)] as acolourless oil. Alkene 19 existed exclusively as the trans C(O)—NProconformer. In addition, restricted rotation about the N—CO carbamatebond was also observed resulting in a 1:1 mixture of conformers: [α]_(D)−118.2 (c 0.34 in CH₂Cl₂); δ_(H) (400 MHz; CDCl₃; Me₄Si) 1.46 [9H, s,C(CH₃)₃], 1.74 (1H, dd, J 11.8 and 5.7, 10-H_(A)H_(B)), 1.93 (1H, dd, J12.5 and 6.5, 11-H_(A)H_(B)), 2.11-2.45 (4H, m, 8-H₂, 10-H_(A)H_(B) and11-H_(A)H_(B)), 3.53 (1H, dd, J 15.7 and 8.0, 5-H_(A)H_(B)), 4.13-4.37(4H, m, 3-H₂, 9-H and 12-H), 4.50 (1H, d, J 15.4, 5-H_(A)H_(B)),5.12-5.29 (2H, m, OCH₂Ph), 5.54-5.82 (2H, m, 6-H and 7-H) and 7.31-7.43(5H, m, Ph); δ_(C) (100 MHz; CDCl₃) 26.7 (CH₂, 11-C), 27.8 [CH₃,C(CH₃)₃], 34.0 (CH₂), 34.2 (CH₂), 34.3 (CH₂), 34.4 (CH₂), 43.9 (CH₂,5-C), 44.5 (CH₂, 5-C), 48.5 (CH₂, 3-C), 49.5 (CH₂, 3-C), 57.2 (CH, 9-C),57.6 (CH, 9-C), 60.1 (CH, 12-C), 67.6 (CH₂, OCH₂Ph), 81.2 [quat.,C(CH₃)₃], 126.8 (CH, 6-C), 126.9 (CH, 6-C), 128.0 (CH, Ph), 128.4 (CH,Ph), 128.7 (CH, Ph), 129.9 (CH, 7-C), 136.3 (quat., Ph), 136.4 (quat.,Ph), 155.7 (quat., NCO₂), 167.8 (quat., 2-C), 167.9 (quat., 2-C) and170.7 (quat., 12-CO); m/z (EI+) 414.2157 (M⁺. C₂₃H₃₀N₂O₅ requires414.2155).

(9S,12S)-1,4-Diaza-4-tert-butyloxycarbonyl-2-oxo-bicyclo[7.3.0]dodecyl-12-carboxylicacid 20

To a stirred solution of alkene 19 (0.11 g, 0.263 mmol) intetrahydrofuran (4 cm³) was added platinum oxide (0.006 g, 0.026 mmol)under a flow of nitrogen. The mixture was hydrogenated (1 atm. ofhydrogen) for 16 h, filtered over Celite™, and the solvent removed invacuo. The residue was dissolved in a solution of 30% hydrobromic acidin acetic acid (3 cm³) and stirred at room temperature for 2 h. Removalof the volatiles in vacuo at 40° C. followed by repeated evaporationfrom methanol:water (3:1) yielded the hydrobromide salt which wasdissolved in a solution of saturated sodium hydrogen carbonate (3 cm³).Dioxane (2 cm³) and di-tert-butyl dicarbonate (0.07 g, 0.32 mmol) wereadded and the resultant suspension was stirred for 24 h, then furtherdi-tert-butyl dicarbonate (0.07 g, 0.32 mmol) was added and stirringcontinued for a further 48 h. Water (10 cm³) was added, the solutionwashed with dichloromethane and the aqueous layer was acidified with 2 Maqueous hydrochloric acid and extracted with dichloromethane. Thecombined organic layers were dried (Na₂SO₄), filtered and the solventremoved to yield an oil (0.072 g) that was purified by chromatography(SiO₂, 1:1:0.3, hexanes-ethyl acetate:acetic acid) to give acid 20 (0.05g, 58%) as a colourless oil. Acid 20 existed exclusively as the trainsC(O)—NPro conformer. In addition, restricted rotation about the N—COcarbamate bond was also observed resulting in a 72:28 mixture ofconformers: [α]_(D) +3.54.4 (c 0.23 in CH₂Cl₂); δ_(H) (400 MHz; CDCl₃;Me₄Si) 1.30-1.48 [11H, m, C(CH₃)₃, 8-H_(A)H_(B) and 11-H_(A)H_(B)],1.63-1.79 (5H, m, 8-H_(A)H_(B), 6-H₂ and 7-H₂), 2.01-2.07 (1H, m,11-H_(A)H_(B)), 2.12-2.32 (2H, m, 10-H₂), 2.62-2.85 (1H, m,5-H_(A)H_(B)), 3.73-3.83 (1.28H, 3-H_(A)H_(B) and 5-H_(A)H_(B)*), 4.0(0.72H, br d, J 14.2, 5-H_(A)H_(B)), 4.20-4.32 (1H, m, 9-H), 4.41*(0.28H, d, J 8.0, 12-H), 4.50 (0.72H, d, J 8.8, 12-H), 4.57 (0.72H, d, J17.8, 3-H_(A)H_(B)), 4.69* (0.28H, d, J 16.5, 3-H_(A)H_(B)) and 8.40(1H, br s, OH); δ_(C) (100 MHz; CDCl₃) 22.1 (CH₂, 7-C), 22.6* (CH₂,7-C), 25.1 (CH₂, 11-C), 25.7* (CH₂, 11-C), 26.6 (CH₂, 6-C), 28.1 [CH₃,C(CH₃)₃], 28.3* [CH₃, C(CH₃)₃], 32.5 (CH₂, 10-C), 32.7* (CH₂, 10-C),35.9* (CH₂, 8-C), 36.3 (CH₂, 8-C), 50.3* (CH₂, 5-C), 51.8 (CH₂, 5-C),54.9* (CH₂, 3-C), 55.4 (CH, 9-C), 56.0* (CH, 9-C), 56.3 (CH₂, 3-C),60.5* (CH, 12-C), 60.8 (CH, 12-C), 80.6* [quat., C(CH₃)₃], 81.0 [quat.,C(CH₃)₃], 155.2 (quat., NCO₂), 155.5* (quat., NCO₂), 169.5* (quat.,2-C), 169.8 (quat., 2-C), 174.9 (quat., 12-CO) and 175.1* (quat.,12-CO); m/z (EI+) 326.1837 (M⁺. C₁₆H₂₆N₂O₅ requires 326.1842).

(2S,9′S,12′S)-2-{[(1′,4′-Diaza-2′-oxobicyclo[7.3.0]dodecyl)-12′-carbonyl]amino}-1,5-pentanedioicacid trifluoroacetate salt 22

Bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BoP—Cl) (0.057 g, 0.225mmol) was added to a solution of acid 10 (0.05 g, 0.15 mmol), L-glutamicacid di-tert-butyl ester hydrochloride 21 (0.062 g, 0.21 mmol) andtriethylamine (0.061 cm³, 0.44 mmol) in dichloromethane (5 cm³) at 0° C.The mixture was stirred for 17 h, washed with saturated aqueous sodiumhydrogen carbonate, 2 M aqueous hydrochloric acid, dried (Na₂SO₄),filtered and the solvent removed to yield an oil (0.108 g) which waspurified by chromatography (SiO₂, 1:1, 1:2, 1:3, hexane-ethyl acetate)to afford an inseparable mixture (0.074 g) of the desired amidecontaminated with a glutamate/bis(2-oxo-3-oxazolidinyl)phosphinicchloride adduct. The mixture was dissolved in dichloromethane (5 cm³),trifluoroacetic acid (2 cm³) was added and the solution stirred for 4 h.Further trifluoroacetic acid (1 Cm³) was then added and stirringcontinued for 2 h. The volatiles were removed in vacuo, the residuesuspended in water and filtered through a plug of cotton wool. Thefiltrate was subsequently purified by RP-HPLC [water (0.05%trifluoroacetic acid):acetonitrile, 90:10, 13 ml min⁻¹] to affordtrifluoroacetate 22 (0.039 g, 55% from 20) as a white solid aftertrituration from diethyl ether/toluene. Macrocycle 22 existedexclusively as the trans C(O)—NPro conformer: mp 50-100° C.; [α]_(D)−22.3 (c 0.35 in MeOH); δ_(H) (400 MHz; CDCl₃; Me₄Si) 1.68-2.04 (9H, m,7′-H₂, 6′-H₂, 10′-H₂, 3′-H_(A)H_(B), 8′-H_(A)H_(B) and 11′-H_(A)H_(B)),2.19-2.26 (2H, 8′-H_(A)H_(B) and 3′-H_(A)H_(B)), 2.42-2.48 (1H, m,11′-H_(A)H_(B)), 2.50-2.58 (2H, m, 4-H₂), 3.04 (1H, td, J 11.2 and 2.5,5′-H_(A)H_(B)), 3.33 1H, dt, J 14.1 and 4.9, 5′-H_(A)H_(B)), 3.82 (1H,d, J 14.1, 3′-H_(A)H_(B)), 4.32 (1H, d, J 14.0, 3′-H_(A)H_(B)),4.38-4.45 (2H, m, 9′-H and 2-H) and 4.59 (1H, dd J 9.6 and 1.8, 12′-H);δ_(C) (100 MHz; CDCl₃) 25.6 (CH₂, 6′-C or 7′-C), 26.0 (CH₂, 6′-C or7′-C), 28.1 (CH₂, 3-C), 29.9 (CH₂, 11′-C), 32.3 (CH₂, 4-C), 34.0 (CH₂,10′-C or 8′-C), 35.2 (CH₂, 10′-C or 8′-C), 46.9 (CH₂, 5′-C), 48.7 (CH₂,3′-C), 54.3 (CH, 2-C), 62.9 (CH, 12′-C), 64.6 (CH, 9′-C), 118.6 (quat.,q, J 291, CF₃), 165.1 (quat., q, J 36.2, CF₃CO₂H), 167.9 (quat., 2′-C),175.9 (quat., 12′-CO), 177.1 (quat., 1-C), and 179.4 (quat., 5-C); m/z(FAB+) 356.1824 [MH(free base)⁺. C₁₆H₂₆N₃O₆ requires 356.1822].

Example 3 Synthesis of (3S,8R,10S,13S) 3-amino-1,11-diaza-8,10-carboxy2,12-oxobicyclo[13.3.0]hexadecane trifluoroacetate

N-Benzyl-L-proline hydrochloride 24 (BP)

A suspension L-proline 23 (6.0 g, 52.1 mmol) and potassium hydroxide(85%, 11.1 g, 198 mmol) in isopropanol (50 cm³) was heated at 40° C.until mostly dissolved. Benzyl chloride (9.2 cm³, 79.7 mmol) was addeddropwise during which the internal temperature rose to 70° C. Theaddition was stopped, the reaction flask placed in an ice bath until aninternal temperature of 40° C. had been obtained, and then the additioncontinued at 40° C. The milky-white suspension was stirred at 40° C. for5.5 h, cooled to room temperature and neutralised with 32% hydrochloricacid until pH=4 had been obtained. Chloroform (50 cm³) was added, thesuspension stored overnight at 0° C. and the white solid was removed byfiltration. The filtrate was concentrated in vacuo, suspended inacetone, the solid filtered and air dried to give BP 24 (9.842 g, 78%)as a white solid: mp 173-176° C. (dec) (lit., 174-175, free base)[α]_(D) −22.9 (c 0.20 in H₂O) (lit.,¹ −25.8 c 1 in EtOH, free base);δ_(H) (300 MHz; D₂O) 1.91-2.21 (3H, Proγ-H₂, Proβ-H_(A)H_(B)), 2.47-2.60(1H, m, Proβ-H_(A)H_(B)), 3.27-3.36 (1H, m, Proδ-H_(A)H_(B)), 3.60-3.67(1H, m, Proδ-H_(A)H_(B)), 4.14 (1H, dd, J 9.3 7.0, Proα-H), 4.37 (1H, d,J 12.9, NCH_(A)H_(B)Ph), 4.44 (1H, d, J 12.9, NCH_(A)H_(B)Ph) and7.45-7.54 (5H, m, Ph); δ_(C) (75 MHz; D₂O) 22.4 (CH₂, Proγ-C), 28.4(CH₂, Proβ-C), 54.6 (CH₂, Proδ-C), 58.3 (CH₂, NCH₂Ph), 67.1 (CH,Proα-C), 129.0, 130.4 (CH, Ph), 129.6 (quat., Ph), 129.9 (CH, Ph) and172.4 (quat., Proα-CO).

(S)-2-[N-(N′-Benzylprolyl)amino]benzophenone 25 (BPB)

To an opaque solution of BP 24 (10.567 g, 43.84 mmol) and triethylamine(5.9 cm³, 43.84 mmol) in dry dichloromethane (70 cm³) was added thionylchloride (4.3 cm³, 58.5 mmol) over 5 min at −30° C. The resultant orangesuspension was allowed to warm to −10° C. over 30 min, stirred for 30min at this temperature and cooled to −30° C. A solution of2-aminobenzophenone (5.76 g, 29.2 mmol) in dry dichloromethane (30 cm³)was added over 5 min and the yellow-orange solution stirred at roomtemperature overnight. The mixture was cooled to 0° C. and a solution ofsodium carbonate (9.1 g) in water (50 cm³) was added carefully. Afterthe evolution of gas had ceased, the organic layer was removed, theaqueous layer extracted with dichloromethane, and combined extractsdried (MgSO₄). Evaporation of the solvent and initial purification bychromatography (silica gel, hexanes:ether, 4:1, 3:1, 1:1) [to removemost of 2-aminobenzophenone] gave a solid (6.405 g) which by ¹H NMRcontained 10% 2-aminobenzophenone. Recrystallisation from boilinghexanes afforded BPB 25 (5.218 g, 46%) as yellow crystals: mp 99-102° C.(lit., 101-102° C.); [α]_(D) −129.1 (c 0.12 in MeOH) (lit., −134 c 1 inMeOH); δ_(H) (300 MHz; CDCl₃; Me₄Si) 1.78-2.04 (3H, Proγ-H₂,Proβ-H_(A)H_(B)), 2.21-2.47 (2H, m, Proβ-H_(A)H_(B), Proδ-H_(A)H_(B)),3.22-3.24 (1H, m, Proδ-H_(A)H_(B)), 3.34 (1H, dd, J 10.1 and 4.6,Proα-H), 3.61 (1H, d, J 12.9, NCH_(A)H_(B)Ph), 3.94 (1H, d, J 12.9,NCH_(A)H_(B)Ph), 7.08-7.65 (12H, m, Ph), 7.81 (2H, d, J 8.2, Ph) and11.6 (1H, s, N—H); δ_(C) (75 MHz; CDCl₃) 24.1 (CH₂, Proγ-C), 30.9 (CH₂,Proβ-C), 53.8 (CH₂, Proδ-C), 59.8 (CH₂, NCH₂Ph), 68.2 (CH, Proα-C),121.4, 122.7 (CH, Ph), 125.2 (quat., Ph), 127.1, 128.1, 128.2, 129.0,130.0, 132.4, 132.5, 133.3 (CH, Ph), 138.0, 138.5, 139.0 (quat., Ph),174.6 (quat., Pro-CON) and 198.0 (quat., Ph₂CO).

Gly-Ni-BPB Complex 26

A solution of potassium hydroxide (4.56 g, 81.3 mmol) in methanol (30cm³) was added to a suspension of BPB 25 (4.46 g, 11.61 mmol), glycine(4.36 g, 58.07 mmol) and nickel nitrate hexahydrate (6.93 g, 23.2 mmol)at 50° C. under an atmosphere of nitrogen. The mixture was heated at 60°C. for 75 min, cooled to room temperature and acetic acid (4.65 cm³,81.3 mmol) added. Water (150 cm³) was then added and the mixture allowedto stand at room temperature for 2 h. Extraction of the product withdichloromethane yielded a blood-red syrup which was purified bychromatography (silica gel, dichloromethane:acetone, 7:1, 6:1, 4:1, 3:1,2:1) to give the crude product which was recrystallised from ethylacetate at −20° C. to give complex 26 (4.377 g, 78%) as a red solid: mp226-228° C. (lit., 208-212° C.); [α]_(D) +2010.6 (c 0.104 in MeOH)(lit., +2006 c 0.1 in MeOH); δ_(H) (400 MHz; CDCl₃; Me₄Si) 2.03-2.20(2H, Proγ-H_(A)H_(B), Proδ-H_(A)H_(B)), 2.38-2.49 (1H, m,Proβ-H_(A)H_(B)), 2.55 (1H, m, Proβ-H_(A)H_(B)), 3.30-3.42 (1H, m,Proγ-H_(A)H_(B)), 3.48 (1H, dd, J 10.7 and 5.4, Proα-H), 3.66-3.75 (3H,m, Proδ-H_(A)H_(B), NCH_(A)H_(B)Ph, Glyα-H_(A)H_(B)), 3.79 (1H, d, J20.1, Glyα-H_(A)H_(B)), 4.49 (1H, d, J 12.9, NCH_(A)H_(B)Ph), 6.71 (1H,t, J 7.2, Ph), 6.81 (1H, dd, J 8.2 and 1.5, Ph), 6.93-7.00 (1H, m, Ph),7.11 (1H, br d, J 7.2, Ph), 7.20-7.24 (1H, m, Ph), 7.32 (1H, t, J 7.5,Ph), 7.44 (2H, t, J 7.52, Ph), 7.51-7.57 (3H, m, Ph), 8.09 (2H, d, J7.4, Ph) and 8.30 (1H, d, J 8.6, Ph); δ_(C) (100 MHz; CDCl₃) 23.6 (CH₂,Proγ-C), 30.6 (CH₂, Proβ-C), 57.4 (CH₂, Proδ-C), 61.2 (CH₂, Glyα-C),63.1 (CH₂, NCH₂Ph), 68.9 (CH, Proα-C), 120.8, 124.2 (CH, Ph), 125.1(quat., Ph), 125.6, 126.2, 128.8, 129.0, 129.3, 129.5, 129.6, 131.6,132.1, 133.1 (CH, Ph), 133.3, 134.5, 142.5 (quat., Ph), 171.5 (quat.,C═N), 177.7 (quat., Glyα-CO) and 181.3 (quat., Pro-CON).

AllylGly-Ni-BPB Complex 27

Powdered sodium hydroxide (0.733 g, 18.34 mmol) and allyl bromide (2.06cm³, 22.0 mmol) was added to a stirred solution of complex 26 (3.6 g,7.33 mmol) in dry acetonitrile (160 cm³) under a nitrogen atmosphere.The mixture was stirred for 2.5 h at room temperature, 0.1M aqueoushydrochloric acid (100 cm³) added and the product extracted withdichloromethane. The combined organic layers were dried (MgSO₄), thesolvent removed and the residue purified by chromatography (silica gel,dichloromethane:acetone, 2:1) to give a mixture of diastereomers (1.468g) and one diastereomer (2.174 g). Both fractions were recrystallisedfrom ethyl acetate at −20° C. to give the desired compound (1.9 g);further compound was obtained from crystallization from the motherliquors (0.766 g) to give allyl complex 27 (2.676 g, 68%) as a red solid(single diastereomer): mp 216-219° C. (lit., 217° C.); δ_(H) (300 MHz;CDCl₃; Me₄Si) 2.0-2.16 (2H, m, Proγ-H_(A)H_(B), Proδ-H_(A)H_(B)),2.36-2.55 [3H, m, Proβ-H_(A)H_(B), CH₂(allyl)], 2.74-2.83 (1H, m,Proβ-H_(A)H_(B)), 3.41-3.61 (4H, m, Proγ-H_(A)H_(B), Proα-H,Proδ-H_(A)H_(B), NCH_(A)H_(B)Ph), 4.01 (1H, dd, J 6.3 and 4.1, Glyα-H),4.42 (1H, d, J 12.6, NCH_(A)H_(B)Ph), 5.17 (1H, d, J 17, ═CH_(A)H_(B)),5.38 (1H, d, J 10, ═CH_(A)H_(B)), 6.36-6.50 (1H, m, C(CH)═CH₂),6.61-6.68 (2H, m, Ph), 6.94 (1H, d, J 7.0, Ph), 7.11-7.54 (8H, m, Ph),8.03 (2H, d, J 7.4, Ph) and 8.17 (1H, d, J 8.7, Ph); δ_(C) (75 MHz;CDCl₃) 23.3 (CH₂, Proγ-C), 30.8 (CH₂, Proβ-C), 38.5 [CH₂, CH₂(allyl)],56.9 (CH₂, Proβ-C), 63.1 (CH₂, NCH₂Ph), 70.27, 70.34 (CH, Proα-C,Glyα-C), 119.7 (CH₂, ═CH₂), 120.6, 123.6 (CH, Ph), 126.4 (quat., Ph),127.0, 127.7, 128.8, 128.9, 129.0, 129.8, 131.5, 132.1, 132.2, (CH,C(H)═CH₂, Ph), 133.2, (quat., Ph), 133.3 (CH, Ph), 133.9, 142.5 (quat.,Ph), 170.8 (quat., C═N), 178.8 (quat., Glyα-CO) and 180.3 (quat.,Pro-CON).

(S)-Allylglycine 28

A mixture of allyl complex 27 (2.520 g, 4.69 mmol) and 2M aqueoushydrochloric acid (54 cm³) in methanol (78 cm³), was heated (oil bath80° C.) for 1 h. The light green solution was cooled to room temperatureand 28% ammonia solution was added until pH=9-10. The aqueous layer wasextracted with dichloromethane to recover BPB (1.82 g, 100% recovery)and the aqueous layer was concentrated in vacuo to afford a blue solidthat was purified by ion exchange chromatography (Dowex 50W×8-100, H₂O,then 5% ammonia solution). The ammonia fractions were combined,evaporated to dryness, suspended in toluene and concentrated in vacuo(2×) to yield a solid that was dried on a freeze drier to giveallylglycine 28 (0.455 g, 84%) as a white powder: mp 195-190° C. (dec.)(lit., 208° C.); [α]_(D) −40.5 (c 0.11 in H₂O) (lit., −42.7 c 4 in H₂O);δ_(H) (400 MHz; D₂O) 2.62-2.76 [2H, m, CH₂(allyl)], 3.87 (1H, dd, J 6.9and 5.4, Glyα-H), 5.30-5.35 (2H, m, ═CH₂) and 5.58-5.88 (1H, m,C(H)═CH₂); ); δ_(C) (100 MHz; D₂O) 34.7 [CH₂, CH₂(allyl)], 53.8 (CH,Glyα-C), 120.4 (CH₂, ═CH₂), 131.2 (CH, C(H)═CH₂) and 174.0 (quat.,Glyα-CO).

(S)-N-Benzyloxycarboylallylglycine 29

A solution of benzyl chloroformate (0.135 cm³, 0.956 mmol) in dioxane (1cm³) was added to a solution of allylglycine 28 (0.1 g, 0.870 mmol) andsodium carbonate (0.275 g, 2.60 mmol) in water (5 cm³) at 0° C. Thesolution was allowed to warm to room temperature over 2 h, then stirredfor a further 17 h. The aqueous layer was washed with ether, acidifiedwith 31% hydrochloric acid to pH=1 and extracted with ethyl acetate. Thepooled organic layers were dried (MgSO₄) and the solvent removed invacuo to give carbamate 7 (0.114 g, 53%) as an opaque oil. Carbamate 29was shown to be a 74:26 mixture of rotamers: δ_(H) (300 MHz; CDCl₃;Me₄Si) 2.52-2.67 [2H, m, CH₂(allyl)], 4.37° (0.26H, br s, Glyα-H),4.46-4.52 (0.76H, m, Glyα-H), 5.08-5.18 (4H, m, OCH₂Ph, ═CH₂) 5.48(0.74H, d, J 7.9, N—H), 5.69-5.79 (1H, m, C(H)═CH₂), 6.34 (0.26H, br d,J 5.6, N—H), 7.26-7.34 (5H, m, Ph) and 9.46 (1H, br s, OH); δ_(C) (75MHz; CDCl₃) 36.4 [CH₂, CH₂(allyl)], 53.2 (CH, Glyα-C), 53.9* (CH,Glyα-C), 67.2 (CH₂, OCH₂Ph), 67.7* (CH₂, OCH₂Ph), 119.6 (CH₂, ═CH₂),128.1, 128.3, 128.6 (CH, Ph), 131.9 (CH, C(H)═CH₂), 136.1 (quat., Ph),156.1 (quat., NCO₂) and 176.0 (quat., Glyα-CO).

(S)-N-Benzyloxycarboylallylglycyl-L-proline 31

N,N-Dicyclohexylcarbodiimide (0.103 g, 0.504 mmol) was added to asolution of carbamate (0.114 g, 0.457 mmol), proline methyl esterhydrochloride 30 (0.083 g, 0.504 mmol), N-hydroxybenzotriazole (0.068 g,0.504 mmol) and triethylamine (0.071 cm³, 0.504 mmol) in dichloromethane(12 cm³) at 0° C. The mixture was stirred overnight, stored at 0° C. for3 h and filtered thorough Celite™. The reaction mixture was washed with2M aqueous hydrochloric acid, saturated sodium hydrogen carbonatesolution, dried (MgSO₄) and the solvent removed to yield an oil whichwas purified by chromatography (silica gel, hexanes:ethyl acetate, 2:1,1:1) to afford protected dipeptide (0.141 g) that was suspended indioxane (4 cm³). 1M Aqueous sodium hydroxide (1.95 cm³, 1.95 mmol) wasadded and the mixture stirred at room temperature for 24 h. Water (5cm³) was added and the mixture extracted with dichloromethane. Theaqueous layer was acidified with 31% hydrochloric acid and the productextracted with dichloromethane. The organic layers were pooled, washedwith brine, dried (MgSO₄) and the solvent removed to afford an oil(0.152 g) that was purified by chromatography (silica gel, hexane:ethylacetate, 3:1, then hexane:ethyl acetate:acetic acid, 3:1:0.4, 2:1:0.3)to give acid 31 (0.102 g, 64%) as a colourless oil. Acid 31 was shown tobe a 87:13 trans:cis mixture of conformers by ¹H NMR analysis (the ratiowas estimated by integration of multiplets at δ 3.33-3.50 and 3.62-3.77assigned to the Proδ-H₂ atoms of the minor and major conformersrespectively): [α]_(D) −91 (c 0.26 in CH₂Cl₂); δ_(H) (300 MHz; CDCl₃;Me₄Si) 1.65* (0.13H, br s, Proβ-H_(A)H_(B)), 1.84-2.25 (3.87H, m,Proβ-H_(A)H_(B),* Proβ-H₂, Proγ-H₂), 2.34-2.53 [2H, m, CH₂(allyl)],3.33-3.50* (0.26H, m, Proδ-H₂), 3.62-3.77 (1.74H, m, Proδ-H₂), 4.30-4.58(2H, m, Proα-H, Glyα-H), 4.93-5.15 (4H, m, OCH₂Ph, ═CH₂) 5.71-5.85 (1H,m, C(H)═CH₂), 6.0 (0.13H, br d, J 8.5, N—H), 6.06* (0.131H, br d, J 8.7,N—H), 7.26-7.31 (5H, m, Ph) and 8.01 (1H, br s, OH); δ_(C) (75 MHz;CDCl₃) 22.1* (CH₂, Proγ-C), 24.8 (CH₂, Proγ-C), 28.6 (CH₂, Proβ-C),31.2* (CH₂, Proβ-C), 36.6 [CH₂, CH₂(allyl)], 37.8* [CH₂, CH₂(allyl)],46.7* (CH₂, Proδ-C), 47.3 (CH₂, Proδ-C), 52.1 (CH, Glyα-C), 52.3* (CH,Glyα-C), 59.2 (CH, Proα-C), 66.6 (CH₂, OCH₂Ph), 67.1* (CH₂, OCH₂Ph),119.1 (CH₂, ═CH₂), 127.9, 128.0, 128.4, 128.5 (CH, Ph), 132.4 (CH,C(H)═CH₂), 136.1* (quat., Ph), 136.3 (quat., Ph), 156.1 (quat., NCO₂),170.8* (quat., Glyα-CO), 171.4 (quat., Glyα-CO) and 174.6 (quat.,Proα-CO); m/z (EI+) 346.1527 (M⁺. C₁₈H₂₂N₂O₅ requires 346.1529).

(S)-N-Benzyloxycarboylallylglycyl-L-prolyl-L-γ(R)-allylglutamic aciddibenzyl ester 34

Alkene 32 (0.217 g, 0.464 mmol) was dissolved in dichloromethane (7cm³), cooled to 0° C. and trifluoroacetic acid (1.5 cm³) was added.After stirring for 2 h the volatiles were removed in vacuo to yieldtrifluoroacetate 33 as an off white solid. To an ice cold solution ofacid 31 (0.134 g, 0.387 mmol) and triethylamine (0.062 cm³, 0.461 mmol)in dichoromethane (10 cm³) was added dropwise ethyl chloroformate (0.045cm³, 0.464 mmol). The solution was stirred at 0° C. for 40 min then anice cold solution of 33 and triethylamine (0.062 cm³, 0.464 mmol) indichloromethane (5 cm³) was added dropwise and the mixture stirredovernight. The solution was subsequently washed with 2M aqueoushydrochloric acid, saturated sodium hydrogen carbonate solution, dried(MgSO₄) and the solvent removed to yield an oil which was purified bychromatography (silica gel, hexane:ethyl acetate, 2:1) to give protectedtripeptide 34 (0.162 g, 60%) as a colourless oil. Tripeptide 34 wasshown to be a 89:11 trans:cis mixture of conformers by ¹H NMR analysis(the ratio was estimated by integration of the doubles at δ 8.04 and7.10 assigned to the GluN—H atoms of the minor and major conformersrespectively): [α]_(D) −31.2 (c 0.462 in CH₂Cl₂); δ_(H) (400 MHz; CDCl₃;Me₄Si) 1.90-1.99 (2H, m, Proβ-H_(A)H_(B), Proγ-H_(A)H_(B)), 2.06-2.13(3H, m, Proγ-H_(A)H_(B), Gluβ-H₂), 2.19-2.26 (1H, m, Proβ-H_(A)H_(B)),2.33-2.42 [3H, m, CH₂(allyl), CH_(A)CH_(B)(allyl)], 2.47-2.54 (1H, m,CH_(A)CH_(B)(allyl)], 2.63 (1H, p, J 6.7, Gluγ-H), 3.56-3.72 (2H, m,Proδ-H₂), 4.18* (0.11H, q, J 6.2, Glyα-H), 4.38* (0.11H, d, J 7.7,Proα-H), 4.54-4.66 (2.78H, Glyα-H, Proα-H), Gluα-H), 4.86-5.18 (10H, m,3×OCH₂Ph, 2×═CH₂), 5.60-5.81 (3H, m, 2×C(H)═CH₂, GlyN-H), 7.10 (0.89H,d, J 7.4, GluN—H), 7.26-7.39 (15H, m, Ph) and 7.10* (0.11H, d, J 7.9,GluN—H); δ_(C) (100 MHz; CDCl₃) 22.0* (CH₂, Proγ-C), 24.9 (CH₂, Proγ-C),27.5 (CH₂, Proβ-C), 31.3* (CH₂, Proβ-C), 31.4* (CH₂, Gluβ-C), 32.9 (CH₂,Gluβ-C), 35.65* [CH₂, CH₂(allyl)], 35.71* CH₂, CH₂(allyl)], 35.9 [CH₂,CH₂(allyl)], 36.9 [CH₂, CH₂(allyl)], 41.0 (CH, Gluγ-C), 41.9* (CH,Gluγ-C), 46.9* (CH₂, Proδ-C), 47.3 (CH₂, Proδ-C), 50.7 (CH, Gluα-C),51.3* (CH, Gluα-C), 51.8 (CH, Glyα-C), 52.4* (CH, Glyα-C), 59.9 (CH,Proα-C), 60.8* (CH, Proα-C), 66.31* (CH₂, OCH₂Ph), 66.4 (CH₂, OCH₂Ph),66.8 (CH₂, OCH₂Ph), 67.0* (CH₂, OCH₂Ph), 67.2 (CH₂, OCH₂Ph), 117.7,119.0, 119.8* (CH₂, ═CH₂), 127.9, 128.0, 128.14, 128.22, 128.24, 128.31,128.42, 128.46, (CH, Ph), 131.7,* 132.2, 134.2 (CH, C(H)═CH₂), 135.2,135.3,* 135.7, 136.0,* 136.2 (quat., Ph), 155.8 (quat., NCO₂), 156.2*(quat., NCO₂), 170.7* (quat., CO), 170.9 (quat., CO) 171.2 (quat., CO),171.5 (quat., CO), 171.6* (quat., CO), 174.3* (quat., Gluγ-CO) and 174.4(quat., Gluγ-CO); m/z (EI+) 695.3170 (M⁺. C₄₀H₄₅N₃O₈ requires 695.3207).

(3S,8R,10S,13S)3-Amino-1,11-diaza-8,10-carboxy-2,12-oxobicyclo[13.3.0]hexadecanetrifluoroacetate 36

To a solution of protected tripeptide 34 (0.041 g, 0.0589 mmol) in drydichloromethane (7.5 cm³) was addedbis(tricyclohexylphosphine)-benzylidineruthenium dichloride (0.015 g,0.0126 mmol) under an atmosphere of nitrogen. The purple solution washeated at reflux for 48 h, cooled to room temperature and dimethylsulphoxide (0.045 cm³, 0.63 mmol) added. The orange-brown solution wasstirred for 24 h, filtered through a short plug of silica gel (elutingwith hexanes:ethyl acetate, 1:3), and purified by chromatography (C₁₈ RPsilica, water:acetonitrile, 20%-70%) to give the metathesis product 35(48 mg) and other unidentified product(s) as a yellow oil, [m/z (FAB+)668.2977 (MH⁺. C₃₈H₄₂N₃O₈ requires 668.2972]. Metathesis product wasdivided into 2 samples (˜25 mg each); the first sample was dissolved intetrahydrofuran/water (4:1, 5 cm³) and 10 wt. % palladium on activatedcarbon (0.008 g, 0.00749 mmol) was added under an atmosphere ofnitrogen. To the reaction flask was fitted a balloon of hydrogen andstirred for 21 h at room temperature. The reaction mixture was filteredthrough a pad of Celite™, washed with methanol/water (4:1) andconcentrated in vacuo to yield a film. The second sample was dissolvedin tetrahydrofuran (3 cm³) and platinum (IV) oxide (0.00078 g, 0.0034mmol) added was added under an atmosphere of nitrogen. To the reactionflask was fitted a balloon of hydrogen and stirred for 16 h at roomtemperature. The reaction mixture was filtered through a pad of Celite™,washed with methanol: and concentrated in vacuo to yield a film that wasdissolved in methanol:water (4:1, 5 cm³) and 10 wt. % palladium onactivated carbon (0.008 g 0.00749 mmol) was added under an atmosphere ofnitrogen. To the reaction flask was fitted a balloon of hydrogen andstirred for 5 h at room temperature. The reaction mixture was filteredthrough a pad of Celite™, washed with methanol/water (4:1) andconcentrated in vacuo to yield a film. Both samples were combined,purified by RP HPLC [10% acetonitrile: 90% water (containing 0.05%trifluoroacetic acid) then 80:20, 70:30] and dried on a freeze drier togive 36 (0.016 g, 58% from 34) as a white solid. 36 existed exclusivelyas the trans conformer: mp 150-240° C.; [α]_(D) −39.4 (c 0.0864 inwater); δ_(H) (400 MHz; D₂O), 1.20-1.62 (6H, m, 5-H₂, 14-H₂, 7-H₂),1.81-1.90 (1H, m, 4-H_(A)H_(B)), 2.04-2.36 (7H, m, 4-H_(A)H_(B), 9-H₂,6-H₂, 15-H₂), 2.43 (1H, br t, J 6.2, 8-H), 3.68-3.79 (2H, m, 16-H₂),4.48-4.49 (1H, m, H-3) and 4.51-4.55 (1H, m, 10-H) [13-H obscured byHOD]; δ_(C) (100 MHz; D₂O) 18.2 (CH₂, 5-C), 23.9 (CH₂, 14-C), 24.2 (CH₂,15-C), 26.0 (CH₂, 6-C), 26.8 (CH₂, 4-C), 28.0 (CH₂, 7-C), 30.3 (CH₂,9-C), 38.2 (CH, 8-C), 47.1 (CH₂, 16-C), 50.6 (CH, 10-C), 50.9 (CH, 3-C),59.5 (CH, 13-C), 116.25 (q, J 291.7, CF₃), 162.8 (q, J 35.2, CF₃CO₂H),169.2 (quat., 2-C), 174.0 (quat., 10-CO), and 179.8 (quat., 8-CO); m/z(FAB+) 356.1811 [MH(free base)⁺. C₁₆H₂₆N₃O₆ requires 356.1821].

Example 4 Synthesis of (2S,3′S,8′R)-2-{[(3′-Amino-1aza-2′-oxo-bicylo[6.3.0]undecyl)11′-carbonyl]amino}-1,5-pentandioic acidtrifluroacetate salt

(S)-Ethyl N-tert-butoxycarbonylpyroglutamate 38

(S)-Pyroglutamic acid 9 (3.0 g, 15.5 mmol) was suspended in ethanol (40cm³) and cooled to 0° C. under a nitrogen atmosphere. Thionyl chloride(2.03 g, 17.0 mmol) was added dropwise and the solution was allowed towarm to room temperature overnight. The solution was cooled to 0° C.,neutralised with saturated aqueous sodium hydrogen carbonate andextracted with chloroform. The combined organic layers were pooled,dried (Na₂SO₄), filtered and the solvent removed to give the crude ester37 (2.53 g, 104%) which was used without further purification. To asolution of ester 37 (2.53 g) and dimethylaminopyridine (0.19 g, 1.55mmol) in acetonitrile (30 cm³) was added di-tert-butyl dicarbonate (3.71g, 17.05 mmol). The resultant solution was stirred at room temperaturefor 18 h, concentrated in vacuo and purified by chromatography (SiO₂,3:1, 2:1, hexanes-ethyl acetate) to give a yellow solid that wasrecrystallised from hexanes to afford carbamate 38 (3.35 g, 85%, in 2steps) as a white solid: mp 53-54° C. (lit. 53-54° C.); [α]_(D) −39.8 (c0.5 in MeOH) [lit. −46.3 (c 1.5 in MeOH)]; δ_(H) (300 MHz; CDCl₃; Me₄Si)1.26 (3H, t, J 7.1, OCH₂CH₃), 1.48 [9H, s, C(CH₃)₃], 1.96-2.06 (1H, m),2.24-2.68 (3H, m), 4.22 (2H, q, J 7.2, OCH₂CH₃), and 4.58 (1H, dd, J 9.4and 3.0, 5-H); δ_(C) (75 MHz; CDCl₃) 14.2 (CH₃, OCH₂CH₃), 21.5 (CH₂,4-C), 27.9 [CH₃, C(CH₃)₃], 31.2 (CH₂, 3-C), 58.9 (CH, 5-C), 61.7 (CH₂,OCH₂CH₃), 83.5 [quat., C(CH₃)₃], 149.3 (quat., NCO₂), 171.3 (quat.,5-CO) and 173.2 (quat., 2-C).

N-tert-Butoxycarbonyl-(S)-allylglycine-5-allyl-L-proline ethyl ester 43

To a stirred solution of carbamate 38 (1.0 g, 3.89 mmol) in drytetrahydrofuran (40 cm³) was added a 1 mol L⁻¹ tetrahydrofuran solutionof lithium triethylborohydride (4.66 cm³, 4.66 mmol) at −78° C. under atatmosphere of nitrogen. The solution was stirred for 1 h, saturatedaqueous sodium hydrogen carbonate (10 cm³) was added and the coolingbath removed. The temperature was allowed to reach 0° C. then 30%aqueous hydrogen peroxide (35 drops) was added and stirring wascontinued at 0° C. for 50 min. The aqueous layer was extracted withether, the combined organic extracts were washed with brine, dried(MgSO₄), filtered and the solvent removed to give alcohol 39 (1.301 g).Crude alcohol 39 (0.363 g, ca. 1.40 mmol) was dissolved in drydichloromethane (10 cm³), allyltributylstannane (0.87 cm³, 2.80 mmol)was added and the solution cooled to −78° C. under a nitrogenatmosphere. Boron trifluoride etherate (0.36 cm³, 2.80 mmol) was addeddropwise, and the solution was stirred at −78° C. for 2 h, thensaturated aqueous sodium hydrogen carbonate (10 cm³) was added and thereaction mixture warmed to room temperature. The aqueous layer wasextracted with dichloromethane and the pooled organic extracts dried(Na₂SO₄) and filtered. Removal of the solvent in vacuo afforded an oil(1.28 g) which was purified by chromatography (SiO₂, 16:1, 10:1, 8:1,6:1, 4:1, 3:1, hexanes-ethyl acetate) to afford alkene 40 [0.217 g, 54%,in 2 steps (74% brsm)] as a colourless oil. Alkene 40 was shown to be a66:33 mixture of C(1)/C(5) cis:trans isomers together with minorcomponent(s). Due to the complex nature of the NMR spectrum thiscompound was not characterized and used as such for the next step.

Trifluoroacetic acid (2 cm³) was added to a solution of alkene 40 (0.22g, 0.77 mmol) in dichloromethane (4 cm³) and the solution was stirred atroom temperature for 4 h, then the volatiles removed in vacuo to yieldthe trifluoroacetate salt 41 as an oil. Half of this material (0.383mmol) was dissolved in dichloromethane (7 cm³),N-tert-butoxycarbonyl-(S)-allylglycine 42 (0.07 g, 0.32 mmol) andtriethylamine (0.042 g, 0.42 mmol) were added and the solution cooled to0° C. N,N′-Dicyclohexylcarbodiimide (0.065 g, 0.316 mmol) was added, themixture stirred overnight, then filtered through Celite™ to removedicyclohexyl urea. The filtrate was subsequently washed with saturatedaqueous sodium hydrogen carbonate, 2 M aqueous hydrochloric acid, dried(MgSO₄), filtered and the solvent removed to yield an oil (0.113 g)which was purified by chromatography (SiO₂, 4:1, hexane-ethyl acetate)to afford diene 43 (0.064 g, 55%, 2 steps) as a colourless oil. Diene 43was an inseparable mixture of diastereomers [C2/C5, cis/trans, 77:23]:δ_(H) (400 MHz; CDCl₃; Me₄Si) 1.21-1.26 (3H, m, OCH₂CH₃), 1.37 [6.93H,s, C(CH₃)₃], 1.41 [2.07H, s, C(CH₃)₃], 1.64-1.97 (4H, m, Proβ-H₂ andProγ-H₂), 2.0-2.52 [3.77H, m, 2×CH₂(allyl)], 2.75-2.95 [0.23H, m,CH_(A)CH_(B)(allyl)], 4.07-4.21 (2.23H, m, OCH₂CH₃ and Proα-H*),4.32-4.48 (2.77H, Proδ-H, Glyα-H and Proα-H), 4.97-5.26 (5H, m, 2×═CH₂,N—H) and 5.64-5.84 [2H, m, 2×C(H)═CH₂]; δ_(C) (100 MHz; CDCl₃) 13.9*(CH₃, OCH₂CH₃), 14.1 (CH₃, OCH₂CH₃), 26.8 (CH₂, Proβ-C), 28.3 [CH₃,C(CH₃)₃], 29.0* (CH₂, Proγ-C), 29.5 (CH₂, Proγ-C), 37.7* [CH₂,CH₂(allyl)Pro], 37.8 [CH₂, CH₂(allyl)Gly], 38.4* [CH₂, CH₂(allyl)Gly],39.4* [CH₂, CH₂(allyl)Pro], 50.9 (CH, Glyα-C), 51.9* (CH, Glyα-C), 58.2(CH, Proα-C), 58.5* (CH, Proα-C), 59.5 (CH, Proδ-C), 59.8* (CH, Proδ-C),61.0 (CH₂, OCH₂CH₃), 61.9* (CH₂, OCH₂CH₃), 79.4* [quat., C(CH₃)₃], 79.7[quat., C(CH₃)₃], 117.2* (CH₂, ═CH₂), 118.3 (CH₂, ═CH₂), 118.35 (CH₂,═CH₂), 118.7* (CH₂, ═CH₂), 132.7* (CH, C(H)═CH₂), 133.2 [CH, C(H)═CH₂],134.0 [CH, C(H)═CH₂], 134.7* [CH, C(H)═CH₂], 154.7* (quat., NCO₂), 155.3(quat., NCO₂), 170.5* (quat., CO), 171.4 (quat., CO), 171.6* (quat., CO)and 172.1 (quat., CO); m/z (FAB+) 381.23896 (MH⁺. C₂₀H₃₃N₂O₅ requires381.23895).

(8R,3S,11S)1-Aza-3-(tert-butyloxycarbonylamino)-11-ethoxycarbonyl-2-oxobicyclo[6.3.0]undec-5-ene44

To a degassed solution of diene 43 (0.064 g, 0.170 mmol) in drydichloromethane (43 cm³) was addedbis(tricyclohexylphosphine)benylidineruthenium dichloride (Grubbscatalyst) (0.014 g, 0.0170 mmol) under a nitrogen atmosphere and theresultant purple solution heated at reflux for 24 h. The orange/brownsolution was cooled to room temperature, dimethyl sulphoxide (0.160 cm³,2.26 mmol) was added and the solution stirred overnight. The solvent wasremoved in vacuo and the residue purified by chromatography (SiO₂, 2:1,1:1, hexanes-ethyl acetate) to give alkene 44 (0.044 g, 74%) as acolourless oil. Alkene 44 existed exclusively as the trans C(O)—NProconformer: [α]_(D) −93.2 (c 0.29 in CH₂Cl₂); δ_(H) (400 MHz; CDCl₃;Me₄Si) 1.25 (3H, t, J 7.1, OCH₂CH₃), 1.42 [9H, s, C(CH₃)₃], 1.89-1.98(2H, m, 10-H_(A)H_(B) and 9-H_(A)H_(B)), 2.01-2.09 (1H, m,9-H_(A)H_(B)), 2.11-2.16 (1H, m, 10-H_(A)H_(B)), 2.24-2.33 (1H, m,4-H_(A)H_(B)), 2.42 (1H, dq, J 15.2 and 3.4, 7-H_(A)H_(B)), 2.70-2.80(2H, m, 4-H_(A)H_(B) and 7-H_(A)H_(B)), 4.15 (3H, q, J 7.1, OCH₂CH₃ and8-H obscured), 4.47 (1H, dd, J 8.4 and 2.8, 11-H), 4.84 (3H, br q, J7.8, 3-H), 5.59 (1H, d, J 7.3, N—H), 5.67-5.73 (1H, m, 6-H) and5.77-5.83 (1H, m, 5-H); δ_(C) (100 MHz; CDCl₃) 14.0 (CH₃, OCH₂CH₃), 27.1(CH₂, 10-C), 28.2 [CH₃, C(CH₃)₃], 32.77 (CH₂, 7-C or 9-C), 32.84 (CH₂,9-C or 7-C), 35.2 (CH₂, 4-C), 51.7 (CH, 3-C), 58.6 (CH, 8-C), 60.2 (CH,11-C), 60.8 (CH₂, OCH₂CH₃), 79.4 [quat., C(CH₃)₃], 125.7 (CH, 6-C),129.1 (CH, 5-C), 155.1 (quat., NCO₂), 170.9 (quat., 11-CO) and 171.7(quat., 2-C); m/z (EI+) 352.2001 (M⁺. C₁₈H₂₈N₂O₅ requires 352.1998).

(2S,3S,8′R,11′S)-Di-tert-butyl2-{[1′-aza-3′-(tert-butyloxycarbonylamino)-2′-oxobicyclo[6.3.0]-undec-5-ene]-11-carbonyl]amino}-1,5-pentandioate47

1M Aqueous sodium hydroxide (0.63 cm³, 0.63 mmol) was added to asolution of ester 44 in dioxane (1.3 cm³) and the opaque solutionstirred for 23 h at room temperature. The reaction mixture was dilutedwith water, extracted with dichloromethane and the aqueous layeracidified with solid citric acid and extracted with dichloromethane. Thecombined organic layers were dried (Na₂SO₄), filtered and the solventremoved to yield acid 45 (0.040 g). Hydrochloride 46 (0.048 g, 0.1625mmol) was added to a solution of 45 and the solution then cooled to 0°C. Triethylamine (0.033 cm³, 0.325 mmol) andbis(2-oxo-3-oxazolidinyl)phosphinic chloride (BoP—Cl) (0.041 g, 0.163mmol) were added and the mixture stirred for 21 h. The reaction mixturewas washed with saturated aqueous sodium hydrogen carbonate, 2 M aqueoushydrochloric acid, dried (Na₂SO₄), filtered and the solvent removed toyield an oil (0.078 g) which was purified by chromatography (SiO₂, 1:1,hexane-ethyl acetate) to afford amide 47 (0.050 g, 70%, in 2 steps) as acolourless oil. Amide 47 existed exclusively as the trans C(O)—NProconformer: [α]_(D) −79.4 (c 0.47 in CH₂Cl₂); δ_(H) (400 MHz; CDCl₃;Me₄Si) 1.39-1.50 [27H, m, 3×C(CH₃)₃], 1.78-1.91 (3H, m, 10′-H_(A)H_(B),3-H_(A)H_(B) and 9′-H_(A)H_(B)), 2.0-2.33 (7H, m, 10′-H_(A)H_(B),3-H_(A)H_(B), 4-H₂, 9′-H_(A)H_(B), 7′-H_(A)H_(B) and 4′-H₄H_(B)),2.80-2.95 (2H, br m, 7′-H_(A)H_(B) and 4′-H_(A)H_(B)), 4.15 (1H, br q, J7.1, 8′-H), 4.42 (1H, ddd, J 7.8, 7.8 and 5.2, 2-H), 4.66 (1H, d, J 7.5,11′-H), 4.97 (1H, br q, J 7.4, 3′-H), 5.57 (1H, d, J 7.8, N—H),5.61-5.67 (1H, m, 6′-H), 5.81 (1H, br t, J 7.8, 5′-H and 7.47 (1H, d, J8.0, N—H); δ_(C) (100 MHz; CDCl₃) 26.1 (CH₂, 10′-C), 27.9 [CH₃,C(CH₃)₃], 27.95 [CH₃, C(CH₃)₃], 28.2 [CH₃, C(CH₃)₃], 28.1, (CH₂, 3-C),31.3 (CH₂, 4-C), 32.0 (CH₂, 7′-C), 33.2 (CH₂, 9′-C), 35.7 (CH₂, 4′-C),51.0 (CH, 3′-C), 51.9 (CH, 2-C), 58.6 (CH, 8′-C), 60.8 (CH, 11′-C), 79.6[quat., C(CH₃)₃], 80.4 [quat., C(CH₃)₃], 81.9 [quat., C(CH₃)₃], 124.9(CH, 6′-C), 130.4 (CH, 5′-C), 155.0 (quat., NCO₂), 169.9 (quat., 1-C),170.5 (quat., 11′-CO), 171.8 (quat., 2′-C or 5-C) and 171.9 (quat., 2′-Cor 5-C); m/z (FAB+) 566.3454 [MH⁺. C₂₉H₄₈N₃O₈ requires 566.3441].

(2S,3′S,8′R,11′S)2-{[(3′-Amino-1′-aza-2′-oxobicyclo[6.3.0]-undecyl)-11′-carbonyl]amino}-1,5-pentanedioicacid trifluoroacetate salt 48

PtO₂ (0.00184 g, 0.0081 mmol) was added to a stirred solution of amide47 (0.046 g, 0.081 mmol) in tetrahydrofuran (4 cm³) under a nitrogenatmosphere. The mixture was hydrogenated (1 atm. of hydrogen) overnight,filtered through Celite™, and the solvent removed in vacuo. The residuewas dissolved in dichloromethane (5 cm³), trifluoroacetic acid (3 cm³)added and the solution stirred for 5 h at room temperature. Removal ofthe volatiles in vacuo, purification by RP HPLC [10% acetonitrile:90%water (containing 0.05% trifluoroacetic acid)] and trituration fromether/toluene gave 13 (0.0228 g, 60%, 2 steps) as a hygroscopicoff-white solid. 48 existed exclusively as the trans C(O)—NProconformer: mp 50-120° C.; [α]_(D) −27.9 (c 0.33 in MeOH); δ_(H) (400MHz; D₂O) 1.37-1.85 (8H, m, 5′-H₂, 6′-H₂, 4′-H₂ and 9′-H₂), 1.98-2.28(5H, m, 3-H₂, 7′-H₂ and 10′-H_(A)H_(B)), 2.36 (1H, dt, J 12.4 and 7.2,10′-H_(A)H_(B)), 2.57 (2H, t, J 7.6, 4-H₂), 4.23 (1H, br t, J 8.2, 8′-H)and 4.41 (3H, m, 2-H, 11′-H, 3′-H); δ_(C) (100 MHz; D₂O) 21.6 (CH₂, 5′-Cor 6′-C), 24.2 (CH₂, 5′-C or 6′-C), 25.5 (CH₂, 3-C), 27.1 (CH₂, 10′-C),29.7 (CH₂, 4-C), 31.5 (CH₂, 9′-C), 32.8 (CH₂, 7′-C), 35.0 (CH₂, 4′-C),51.2 (CH, 3′-C), 51.9 (CH, 2-C), 59.9 (CH, 8′-C), 61.4 (CH, 11′-C),116.2 (quat., q, J 291, CF₃), 162.7 (quat., q, J 35.2, CF₃CO₂H), 168.5(quat., 2′-C), 173.7 (quat., 11′-CO), 174.7 (quat., 1-C) and 176.8(quat., 5-C); m/z (FAB+) 356.1816 [MH(free base)⁺. C₁₆H₂₆N₃O₆ requires356.1822].

Example 5 Synthesis of(9R,11S,14S)-1,4,12-triaza-9,11-carboxy-2,13-dioxobicyclo[14.3.0]heptadecanetrifluoroacetate

N-tert-Butyloxycarbonyl-L-glutamic acid dibenzyl ester 50

Triethylamine (2.25 cm³, 2.2 mmol) was added to a stirred solution ofL-glutamic acid dibenzyl ester p-toluenesulphonate 49 (4.0 g, 8.01 mmol)in dichloromethane (80 cm³) at 0° C. Di-tert-butyldicarbonate (2.1 g,9.61 mmol) was then added and the solution stirred for 19 h. The solventwas removed in vacuo, the residue dissolved in the minimum amount ofdichloromethane and washed sequentially with 2M aqueous hydrochloricacid, saturated aqueous sodium hydrogen carbonate and dried (MgSO₄).Removal of the solvent yielded a white solid (3.42 g) which was purifiedby crystallisation from ether/hexanes to give carbamate 50 (3.321 g,97%) as a white solid: mp 73-76° C.; δ_(H) (400 MHz; CDCl₃; Me₄Si) 1.46[9H, s, C(CH₃)₃], 1.96-2.07 (1H, m), 2.22-2.27 (1H, m), 2.38-2.52 (2H,m), 4.40 (1H, br d, J 3.6, Gluα-H), 5.12-5.19 (5H, m, 2×OCH₂Ph, Gluα-NH)and 7.29-7.39 (10H, m, 2×Ph) δ_(C) (100 MHz; CDCl₃) 27.6 (CH₂, Gluβ-C),28.1 [CH₃, C(CH₃)₃], 30.1 (CH₂, Gluγ-C), 52.8 (CH, Gluα-C), 66.4 (CH₂,OCH₂Ph), 67.1 (CH₂, OCH₂Ph), 79.7 [quat., C(CH₃)₃], 128.1, 128.2, 128.3,128.5, 128.51, (CH, Ph), 135.2, 135.7 (quat., Ph), 155.3 (quat., NCO₂)and 172.0, 172.4 (quat., CO).

N-tert-Butyloxycarbonyl-L-γ(R)-allylglutamic acid dibenzyl ester 51

A tetrahydrofuran solution of lithium hexamethyldisilazide (1 mol L⁻¹,10.3 cm³, 10.3 mmol) was added dropwise to a solution of carbamate 50(2.0 g, 4.68 mmol) in dry tetrahydrofuran (20 cm³) at −78° C. under anatmosphere of nitrogen. The resultant yellow solution was stirred atthis temperature for 40 min and allyl bromide (1.31 cm³, 14.0 mmol) wasadded. The solution was stirred for 1.5 h at −78° C. and quenched by theaddition of saturated aqueous ammonium chloride (20 cm³). The coolingbath was removed and the mixture allowed to warm to room temperatureover 1.5 h. A white solid was removed by filtration, washed with ethylacetate and the filtrate concentrated in vacuo to yield an orange oil(2.312 g) which was purified by chromatography (silica gel, 6:1,hexane:ethyl acetate, then 5:1) to give alkene 51 (1.589 g, 73%) as acolourless oil: [α]_(D) +19.4 (c 0.232 in CH₂Cl₂); δ_(H) (300 MHz;CDCl₃; Me₄Si) 1.43 [9H, s, C(CH₃)₃], 2.06 (2H, m, allyl-CH₂), 2.30-2.44(2H, m, Gluβ-H₂), 2.64 (1H, p, J 6.7, Gluγ-H), 4.41 (1H, br d, J 7.6,Gluα-H), 5.01-5.71 (7H, m, 2×OCH₂Ph, ═CH₂, Gluα-NH), 5.60-5.74 (1H, m,C(H)═CH₂) and 7.31-7.35 (10H, m, 2×Ph); δ_(C) (75 MHz; CDCl₃) 28.2 [CH₃,C(CH₃)₃], 33.4 (CH₂, allyl-CH₂), 36.2 (CH₂, Gluβ-C), 41.7, (CH, Gluγ-C),52.2 (CH, Gluα-C), 66.4 (CH₂, OCH₂Ph), 67.0 (CH₂, OCH₂Ph), 80.0 [quat.,C(CH₃)₃], 117.6 (CH₂, ═CH₂), 128.16, 128.18, 128.3, 128.44, 128.47, (CH,Ph), 134.1 (CH, C(H)═CH₂), 135.2, 135.7 (quat., Ph), 155.3 (quat.,NCO₂), 172.1 (quat., Gluα-CO) and 174.6 (quat., Gluγ-CO); m/z (FAB+)468.2382 (MH⁺. C₂₇H₃₄NO₆ requires 468.2386).

N-Allylglycine ethyl ester 54

A solution of ethyl bromoacetate 52 (3 g, 18.0 mmol) in drytetrahydrofuran (20 cm³) was added dropwise to an ice-cold solution ofallylamine 53 (2.05 g, 36 mmol) in dry tetrahydrofuran (20 cm³) over 3min. The solution was stirred at 0° C. for 2.5 h (a white precipitatewas observed after 2 h), concentrated in vacuo and suspended in diethylether. The white suspension was filtered through Celite™ and the solventremoved to yield the crude product which was purified by chromatography(silica gel, 1:1, hexane:ethyl acetate) to give alkene 54 (1.209 g, 47%)as a light yellow oil: δ_(H) (300 MHz; CDCl₃; Me₄Si) 1.17 (3H, t, J 7.1,OCH₂CH₃), 1.70 (1H, br s, N—H), 3.16 [2H, d, J 5.9, CH₂(allyl)], 3.28(2H, s, CH₂Gly), 4.08 (2H, q, J 7.1, OCH₂CH₃), 4.98-5.12 (2H, m, ═CH₂)and 5.70-5.85 (1H, m, C(H)═CH₂); β_(C) (75 MHz; CDCl₃) 13.9 (CH₃,OCH₂CH₃), 49.7 (CH₂), 51.6 (CH₂), 60.4 (CH₂, OCH₂CH₃), 116.2 (CH₂,═CH₂), 135.9 (CH, C(H)═CH₂) and 172.2 (quat., CO).

N-Allyl-N-benzyloxycarbonylglycine 55

N-Allylglycine ethyl ester 54 was prepared as described above usingethyl bromoacetate (9 g, 53.9 mmol) and allylamine (6.15 g, 107.7 mmol).The crude product (7.054 g) that contained ˜30% ofN-allyl-N-bis(2-carboethyoxyethyl)amine as judged by NMR, was dissolvedin dichloromethane (50 cm³), cooled to 0° C. and triethylamine (6.53 g,64.09 mmol) added. Benzylchloroformate (9.25 g, 54.2 mmol) was addeddropwise over 10 min resulting in a white precipitate. After 2 h themixture was stored overnight at 0° C., filtered and the filtrateconcentrated in vacuo. The residue was subsequently suspended indichloromethane, washed with 2M aqueous hydrochloric acid, dried (MgSO₄)and the solvent removed to reveal an oil (13.65 g) containing thedesired carbamate (70%) and benzyl alcohol (30%) as judged by NMR. Thismixture was dissolved in dioxane/methanol (8:3, 110 cm³), 1M aqueoussodium hydroxide (40 cm³, 40 mmol) added and the solution stirred for 2h at room temperature. Brine (50 cm³) was added and the aqueous layerextracted with diethyl ether. The aqueous layer was acidified with 32%hydrochloric acid and extracted with ethyl acetate. The combined organiclayers were dried (MgSO₄), concentrated in vacuo, and the residuepurified by chromatography (silica gel, hexane:ethyl aceate:acetic acid,3:1:0.4) to give acid 7 (7.05 g, 52%, 3 steps) as a colourless syrup.Acid 55 existed as a 1:1 mixture of carbamate rotamers: δ_(H) (400 MHz;CDCl₃; Me₄Si) 4.02-4.08 [4H, m, CH₂(allyl), CH₂(Gly)], 5.17-5.24 (4H, m,OCH₂Ph, ═CH₂), 5.77-5.84 (1H, m, C(H)═CH₂), 7.32-7.39 (m, 5H, Ph) and8.45 (1H, br s, OH); δ_(C) (100 MHz; CDCl₃) 47.1, 47.7 [CH₂, CH₂(Gly)],50.5, 50.7 [CH₂, CH₂(allyl)], 67.6, 67.7 (CH₂, OCH₂Ph), 117.8, 118.3(CH₂, ═CH₂), 127.7, 127.97, 128.01, 128.4, (CH, Ph), 132.7, 132.8 (CH,C(H)═CH₂) 136.1 (quat., Ph), 155.8, 156.5, (quat., NCO₂) and 174.7,174.9 (quat., CO).

N-Allyl-N-benzyloxycarbonylglycyl-L-proline methyl ester 56

1-(3-Dimethylaminopropyl-3-ethylcarbodiimide hydrochloride (EDCI) (0.453g, 2.36 mmol) was added to a solution of proline methyl esterhydrochloride 30 (0.356 g, 2.15 mmol), acid 55 (0.590 mmol, 2.35 mmol)and triethylamine (0.481 g, 4.73 mmol) in dichloromethane (20 cm³) at 0°C. The resultant solution was stirred for 19 h, washed with 2M aqueoushydrochloric acid and saturated aqueous sodium hydrogen carbonate, dried(MgSO₄) and the solvent removed to yield an oil which was purified bychromatography (silica gel, hexanes:ethyl acetate, 2:1, 1:1) to affordprotected dipeptide 56 (0.508 g, 66%) as a colourless oil. Dipeptide 56was shown to be a 76:24 trans:cis mixture of conformers by ¹³C NMRanalysis (the ratio was estimated by integration of signals at δ 22.0and 24.5, 24.7, assigned to the Proγ-C atoms of the minor and majorconformers respectively). In addition, restricted rotation about theGlyN—CO carbamate bond was also observed resulting in a further 1:1mixture of conformers: [α]_(D) −54.6 (c 1.17 in CH₂Cl₂); δ_(H) (400 MHz;CDCl₃; Me₄Si) 1.81-2.14 (4H, m, Proβ-H₂, Proγ-H₂), 3.29-3.58 (2H, m,Proδ-H₂), 3.64, 3.66, 3.69* (3H, s, OCH₃), 3.84-4.20 [4H, m, CH₂(allyl),Glyα-H₂], 4.40-4.48 (1H, m, Proα-H), 5.07-5.17 (4H, m, OCH₂Ph, ═CH₂),5.69-5.81 (1H, m, C(H)═CH₂) and 7.24-7.30 (m, 5H, Ph); δ_(C) (100 MHz;CDCl₃) 22.0* (CH₂, Proγ-C), 24.62, 24.73 (CH₂, Proγ-C), 28.66, 28.73(CH₂, Proβ-C), 31.1,* 31.3* (CH₂, Proβ-C), 45.93, 45.98 (CH₂, Proδ-C),46.5* (CH₂, Proδ-C), 47.5, 48.0 (CH₂, Glyα-C), 50.0, 50.6 [CH₂,CH₂(allyl)], 50.0,* 50.6* [CH₂, CH₂(allyl)], 52.0, (CH₃, OCH₃), 52.31,*52.5* (CH₃, OCH₃), 58.4,* 58.5* (CH, Proα-C), 58.8 (CH, Proα-C), 67.2,67.3 (CH₂, OCH₂Ph), 117.0, 117.5 (CH₂, ═CH₂), 127.5, 127.7, 127.8,128.3, (CH, Ph), 133.2, 133.4 (CH, C(H)═CH₂) 136.4, 136.5 (quat., Ph),155.8, 156.3, (quat., NCO₂), 167.1 (quat., Gly-CO), 167.4,* 167.5*(quat., Gly-CO). 172.0,* 172.2* (quat., Pro-CO) and 172.3, 172.4 (quat.,Pro-CO); m/z (EI+) 360.1676 (M⁺. C₁₉H₂₄N₂O₅ requires 360.1685).

N-Allyl-N-benzyloxycarbonylglycyl-L-proline 57

To a solution of protected dipeptide 56 (0.474 g, 1.31 mmol) in dioxane(13 cm³) was added 1M aqueous sodium hydroxide (6.71 cm³, 6.71 mmol) andthe mixture stirred at room temperature for 20 h. Water (10 cm³) wasadded and the mixture extracted with dichloromethane. The aqueous layerwas acidified with 10% HCl and the product extracted withdichloromethane. The organic layers were pooled, dried (MgSO₄) and thesolvent removed to afford an oil (0.456 g) contaminated with2-hydroxy-1,4-dioxane. Subsequent purification by chromatography (silicagel, hexanes:ethyl acetate, 1:1, 1:2, 1:3) gave acid 57 (0.250 g, 55%)as a colourless oil: Acid 57 was shown to be a 86:14 trans:cis mixtureof conformers by ¹³C NMR analysis (the ratio was estimated byintegration of signals at δ 22.1 and 24.6, 24.7, assigned to the Proγ-Catoms of the minor and major conformers respectively). In addition,restricted rotation about the GlyN—CO carbamate bond was also observedresulting in a further 1:1 mixture of conformers [α]_(D) −117 (c 0.8 inCH₂Cl₂); δ_(H) (300 MHz; CDCl₃; Me₄Si) 1.88-2.25 (4H, m, Proβ-H₂,Proγ-H₂), 3.30-3.69 (2H, m, Proδ-H₂), 3.75-4.26 [4H, m, CH₂(allyl),Glyα-H₂], 4.50-4.58 (1H, m, Proα-H), 5.10-5.22 (4H, m, OCH₂Ph, ═CH₂),5.73-5.82 (1H, m, C(H)═CH₂), 7.33-7.34 (m, 5H, Ph) and 7.84 (1H, br s,OH); δ_(C) (75 MHz; CDCl₃) 22.1* (CH₂, Proγ-C), 24.6, 24.7 (CH₂,Proγ-C), 27.9, 28.0 (CH₂, Proβ-C), 31.1,* 31.3* (CH₂, Proβ-C), 46.5(CH₂, Proδ-C), 46.7* (CH₂, Proδ-C), 47.5, 48.1 (CH₂, Glyα-C), 47.7,*48.2* (CH₂, Glyα-C), 50.2, 50.7 [CH₂, CH₂(allyl)], 50.5,* 50.9* [CH₂,CH₂(allyl)], 58.6* (CH, Proα-C), 59.5 (CH, Proα-C), 67.6 (CH₂, OCH₂Ph),117.3, 117.9 (CH₂, ═CH₂), 127.6, 127.9, 128.0, 128.4, (CH, Ph), 133.0,133.2 (CH, C(H)═CH₂) 136.3 (quat., Ph), 155.9, 156.5, (quat., NCO₂),156.7* (quat., NCO₂), 167.8* (quat., Gly-CO), 168.9, 169.0 (quat.,Gly-CO), 173.6, 173.7 (quat., Pro-CO) and 174.4* (quat., Pro-CO); m/z(EI+) 346.1524 (M⁺. C₁₈H₂₂N₂O₅ requires 346.1529).

N-Allyl-N-benzyloxycarbonylglycyl-L-prolyl-L-γ(R)-allylglutamic aciddibenzyl ester 59

Alkene 51 (0.359 g, 0.768 mmol) was dissolved in dichloromethane (10cm³), cooled to 0° C. and trifluoroacetic acid (2 cm³) was added. Afterstirring for 2 h the volatiles were removed in vacuo to yieldtrifluoroacetate 58 as a white solid. To an ice cold solution of acid 57(0.288 g, 0.655 mmol) and triethylamine (0.150 cm³, 0.80 mmol) was addeddropwise ethyl chloroformate (0.075 cm³, 0.786 mmol). The solution wasstirred at 0° C. for 40 min then an ice-cold solution of 58 andtriethylamine (0.150 cm³, 0.80 mmol) in dichloromethane (15 cm³) wasadded dropwise and the mixture stirred overnight. The solution wassubsequently washed with 2M aqueous hydrochloric acid, saturated aqueoussodium hydrogen carbonate, dried (MgSO₄) and the solvent removed toyield an oil which was purified by chromatography (silica gel,hexane:ethyl acetate, 1:1) to give fully protected tripeptide 59 (0.432g, 94%) as a colourless oil. Tripeptide 59 was shown to be a 85:15trans:cis mixture of conformers by ¹³C NMR analysis (the ratio wasestimated by integration of signals at δ 22.1 and 24.9 assigned to theProγ-C atoms of the minor and major conformers respectively). Inaddition, restricted rotation about the GlyN—CO carbamate bond was alsoobserved resulting in a further 1:1 mixture of conformers: [α]_(D) −38.7(c 0.73 in CH₂Cl₂); δ_(H) (300 MHz; CDCl₃; Me₄Si) 1.75-2.33 [8H, m,Proβ-H₂, Proγ-H₂, Gluβ-H₂, CH₂(allyl)], 2.55-2.66 (1H, m, Gluγ-H),3.25-3.63 (m, 2H, Proδ-H₂), 3.77-4.19 [4H, m, Glyα-H₂, CH₂(allyl)],4.32* (0.15H, d, J 7.7, Proα-H), 4.46-4.60 (1.85H, m, Proα-H, Gluα-H),4.99-5.15 (10H, m, 3×OCH₂Ph, 2×═CH₂), 5.57-5.82 (2H, m, 2×C(H)═CH₂) and7.19-7.34 (16H, m, 3×Ph, GluN-H); δ_(C) (75 MHz; CDCl₃) 22.2* (CH₂,Proγ-C), 24.9 (CH₂, Proγ-C), 27.2* (CH₂, Proβ-C), 27.5 (CH₂, Proβ-C),31.8* [CH₂, CH₂(allyl)], 32.7 [CH₂, CH₂(allyl)], 35.9 (CH₂, Gluβ-C),36.0* (CH₂, Gluβ-C), 41.2 (CH, Gluγ-C), 41.9* (CH, Gluγ-C), 46.4 (CH₂,Proδ-C), 46.9* (CH₂, Proδ-C), 47.6,* (CH₂, Glyα-C), 48.3 (CH₂, Glyα-C),48.2,* (CH₂, Glyα-C), 50.4 [CH₂, CH₂(allyl)], 50.7, 50.9 (CH₂, Gluα-C),51.2,* [CH₂, CH₂(allyl)], 51.4* (CH₂, Gluα-C), 59.96* (CH, Proα-C), 60.0(CH, Proα-C), 60.4* (CH, Proα-C), 66.3 (CH₂, OCH₂Ph), 66.4* (CH₂,OCH₂Ph), 66.5 (CH₂, OCH₂Ph), 67.0 (CH₂, OCH₂Ph), 67.1 (CH₂, OCH₂Ph),67.35 (CH₂, OCH₂Ph), 67.4 (CH₂, OCH₂Ph), 117.0 (CH₂, ═CH₂), 117.1* (CH₂,═CH₂), 117.5 (CH₂, ═CH₂), 117.6 (CH₂, ═CH₂), 117.8* (CH₂, ═CH₂) 118.0*(CH₂, ═CH₂), 127.56,* 127.63,* 127.77,* 127.85,* 128.08, 128.13, 128.17,128.35, 128.39, 128.43 (CH, Ph), 133.3 (CH, C(H)═CH₂), 133.5* (CH,C(H)═CH₂), 134.0* (CH, C(H)═CH₂), 134.2* (CH, C(H)═CH₂), 134.3 (CH,C(H)═CH₂), 135.2* (quat., Ph), 135.3 (quat., Ph), 135.7 (quat., Ph),136.4 (quat., Ph), 136.6* (quat., Ph), 155.9* (quat., NCO₂), 156.5,(quat., NCO₂), 168.4 (quat., Gly-CO), 170.9,* 171.0, 171.4, 171.8*(quat., Pro-CON, Gluα-CO), 174.3* (quat., Gluγ-CO) and 174.4 (quat.,Gluγ-CO); m/z (FAB+) 696.3280 (MH⁺. C₄₀H₄₆N₃O₈ requires 696.3285).

(9R,11S,14S)-1,4,12-Triaza-9,11-carboxy-2,13-dioxobicyclo[14.3.0]-heptadecanetrifluoroacetate 61

Freshly sublimed potassium tert-butoxide (0.009 g, 0.0792 mmol) wasadded to a stirred suspension of1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazolium tetrafluoroborate³63 (0.031 g, 0.0792 mmol) in tetrahydrofuran (6 cm³) under an atmosphereof nitrogen. The resultant suspension was stirred for 2 min then asolution of bis(tricyclohexylphosphine)benzylideneruthenium dichloride64 (0.054 g, 0.066 mol) in dry benzene (6 cm³) was added and the purplesolution was heated at 80° C. for 35 min. The dark brown solution wascooled to room temperature and a solution of protected tripeptide 59(0.155 g, 0.22 mmol) in dry benzene (85 cm³) added and the mixtureheated at 45° C. for 65 h. The solvent was removed in vacuo and theresidue purified by chromatography (silica gel, hexane:ethyl acetate,1:1, 1:2, 1:3, 1:4) to give the metathesis product 60 and unidentifiedproduct(s) (0.110 g) as a brown oil: [m/z (FAB+) 668.2982 (MH⁺.C₃₈H₄₂N₃O₈ requires 668.2972]. The mixture was subsequently dissolved inmethanol/water (4:1, 50 cm³) and 10 wt. % palladium on activated carbon(0.035 g, 0.033 mmol) was added under an atmosphere of nitrogen. To thereaction flask was fitted a balloon of hydrogen and the mixture stirredfor 17 h at room temperature. The reaction mixture was filtered througha pad of Celite™, washed with methanol/water (4:1, 100 cm³) andconcentrated in vacuo to yield a film (0.064 g) that was purified by RPHPLC [20% acetonitrile: 80% water (containing 0.05% trifluoroaceticacid)]. Repeated evaporation from a toluene/ether mixture and drying ona freeze drier gave 61 (0.045 g, 58% from 59) as an off white solid. 61was shown to be a 65:35 trans:cis mixture of conformers by ¹H NMRanalysis (the ratio was estimated by integration of signals at δ 2.99and 3.17-3.33 assigned to the 5-H atoms of the minor and majorconformers respectively): mp 120-130° C.; [α]_(D) −12.5 (c 0.104 inH₂O); δ_(H) (300 MHz; D₂O), 1.24-2.68 (13.65H, m, 5-H_(A)H_(B), 6-H₂,7-H₂, 8-H₂, 9-H, 10-H₂, 15-H₂, 16-H₂), 2.99* (0.35H, dt, J 12.9 and 7.3,5-H_(A)H_(B)), 3.17-3.33 (1H, m, 5-H), 3.40* (0.35H, d, J 15.6,3-H_(A)H_(B)), 3.50-3.73 (2H, m, 17-H₂), 3.98* (0.35H, d, J 15.6,3-H_(A)H_(B)), 4.01 (0.65H, d, J 15.6, 3-H_(A)H_(B)), 4.08 (0.65H, d, J15.8, 3-H_(A)H_(B)), 4.44 (0.65H, dd, J 11.6 and 4.3, 11-H), 4.51(0.65H, dd, J 7.5 and 5.0, 14-H) and 4.59-4.64* (0.7H, m, 11-H, 14-H);δ_(C) (75 MHz; D₂O) 21.0, 21.4,* 21.7,* 22.2,* 23.6, 24.8, 25.2, 28.2,31.2,* 31.3, 31.6 (CH₂, 6-C, 7-C, 8-C, 10-C, 15-C, 16-C), 38.4* (CH,9-C), 40.2 (CH, 9-C), 45.5* (CH₂, 3-C, 5-C), 46.9, 47.0 (CH₂, 17-C,5-C), 47.5*, (CH₂, 17-C), 48.2, (CH₂, 3-C), 49.6 (CH, 11-C), 50.5* (CH,11-C), 59.9* (CH, 14-C), 61.3 (CH, 14-C), 116.2 (q, J 291, CF₃), 162.6(q, J 35.5, CF₃CO₂H), 164.4* (quat., 2-C), 164.9 (quat, 2-C), 173.4,*173.4* (quat., 13-C, 11-CO), 173.4, 173.4 (quat., 13-C, 11-CO), 179.3*(quat., 9-CO) and 179.8 (quat., 9-CO); m/z (FAB+) 356.1829 [MH(freebase)⁺. C₁₆H₂₆N₃O₆ requires 356.1822]; (the minor component wastentatively assigned as glycyl-L-prolyl-L-γ-butylglutamic acidtrifluoroacetate 62 and was shown to be a 78:22 trans:cis mixture ofconformers by ¹³C NMR analysis (the ratio was estimated by integrationof the signals at δ 51.2 and 51.8 assigned to the Gluα-C atoms of theminor and major conformers respectively): mp 155-190° C. (dec.); δ_(H)(400 MHz; D₂O), 0.90 (3H, t, J 6.5, Gluγ-CH₂CH₂CH₂CH₃), 1.33 (4H, br s,Gluγ-CH₂CH₂CH₂CH₃), 1.64 (2H, br q, J 7.0, Gluγ-CH₂CH₂CH₂CH₃), 2.03-2.17(5H, m, Gluβ-H₂, Proγ-H₂, Proβ-H_(A)H_(B)), 2.31-2.35 (0.78H, m,Proβ-H_(A)H_(B)), 2.57 (1H, p, J 6.9, Gluγ-H), 3.59-3.75 (2.18H, m,Proδ-H₂, Glyα-H_(A)H_(B)*), 3.98-4.10 (1.82H, Glyα-H₂,Glyα-H_(A)H_(B)*), 4.47 (1H, t, J 7.5, Gluα-H) and 4.51-4.56 (1H, m,Proα-H); δ_(C) (100 MHz; D₂O) 13.0 (CH₃, Gluγ-CH₂CH₂CH₂CH₃), 21.7 (CH₂,Gluγ-CH₂CH₂CH₂CH₃), 21.8* (CH₂, Gluγ-CH₂CH₂CH₂CH₃), 24.0 (CH₂, Proγ-C),28.1 (CH₂, Gluγ-CH₂CH₂CH₂CH₃), 28.2* (CH₂, Gluγ-CH₂CH₂CH₂CH₃), 29.3(CH₂, Proβ-C), 30.9 (CH₂, Gluγ-CH₂CH₂CH₂CH₃), 31.3*, 31.6*, 32.1* (CH₂),32.3 (CH₂, Gluβ-C), 40.1* (CH₂, Glyα-C), 40.3 (CH₂, Glyα-C), 42.0 (CH,Gluγ-C), 43.0* (CH, Gluγ-C), 46.8 (CH₂, Proδ-C), 47.4* (CH₂, Proδ-C),51.2 (CH, Gluα-C), 51.8* (CH, Gluα-C), 59.9* (CH, Proα-C), 60.2 (CH,Proα-C), 115.5 (q, J 291, CF₃), 162.9 (q, J 35.2, CF₃CO₂H), 165.5(quat., Gly-CO), 166.0* (quat., Gly-CO), 173.5* (quat., CO), 174.0(quat., CO), 174.5* (quat., CO), 174.8 (quat., CO), 180.0 (quat.,Gluγ-CO) and 180.2* (quat., Gluγ-CO); m/z (FAB+) 358.1978 [MH(freebase)⁺. C₁₆H₂₈N₃O₆ requires 358.1978].

Example 6 Synthesis of(2S,9′R,12′S)-2-{[(1′,4′-Diaza-2′-oxobicyclo[7.3.0]-dodecyl)-12′-carbonyl]amino}-1,5-pentanedioicacid trifluoroacetate salt

(6Z,9R,12S)-1,4-Diaza-4-benzyloxycarbonyl-12-tert-butoxycarbonyl-2-oxobicyclo[7.3.0]dodec-6-ene65

To a degassed solution of diene 17 (0.11 g, 0.242 mmol) in drydichloromethane (60 cm³) was addedbis(tricyclohexylphosphine)benzylidineruthenium dichloride (Grubbs'scatalyst) (0.020 g, 0.024 mmol) under an atmosphere of nitrogen and theresultant purple solution heated at reflux for 24 h. Furtherbis(tricyclohexylphosphine)benzylidineruthenium dichloride (Grubbs'scatalyst) (0.020 g, 0.024 mmol) was then added, and refluxing continuedfor a further 24 h. The solution was cooled to room temperature,dimethyl sulphoxide (0.16 cm³, 2.26 mmol) was added and the orange/brownsolution stirred for 22 h. The solvent was removed in vacuo and theresidue purified by chromatography (SiO₂, hexanes-ethyl acetate, 3:1,2:1, 1:1) to give an almost colourless oil which was further purified bychromatography (C₁₈ RP silica, 100:0, 9:1, 9:3, 1:1, 1:9,water-acetonitrile) to give alkene 65 (0.046 g, 46%) as a colourlessoil. Alkene 65 existed exclusively as the trans C(O)—NPro conformer. Inaddition, restricted rotation about the N—CO carbamate bond was alsoobserved resulting in a 1:1 mixture of conformers: [α]_(D) −243.9 (c0.27 in CH₂Cl₂); δ_(H) (400 MH; CDCl₃; Me₄Si) 1.49 [9H, s, C(CH₃)₃],1.84-2.34 (5H, m, 8-H_(A)H_(B), 10-H₂ and 11-H₂), 2.98-3.08 (1H, m,8-H_(A)H_(B)), 3.69-3.88 (2H, m, 5-H_(A)H_(B) and 3-H_(A)H_(B)), 4.0(1H, p, J 7.6, 9-H), 4.33-4.61 (3H, m, 5-H_(A)H_(B), 3-H_(A)H_(B) and12-H), 5.10-5.30 (2H, m, OCH₂Ph), 5.51-5.65 (1H, m, 6-H), 5.98-6.09 (1H,m, 7-H) and 7.29-7.38 (5H, m, Ph); δ_(C) (100 MH; CDCl₃) 27.2 (CH₂,11-C), 28.0 [CH₃, C(CH₃)₃], 33.9 (CH₂, 8-C), 35.1 (CH₂, 10-C), 42.2(CH₂, 5-C), 42.3 (CH₂, 5-C), 45.7 (CH₂, 3-C), 45.9 (CH₂, 3-C), 59.5 (CH,9-C), 59.6 (CH, 9-C), 61.2 (CH, 12-C), 61.3 (CH, 12-C), 67.4 (CH₂,OCH₂Ph), 67.5 (CH₂, OCH₂Ph), 81.4 [quat., C(CH₃)₃], 81.5 [quat.,C(CH₃)₃], 125.8 (CH, 6-C), 126.0 (CH, 6-C), 127.7 (CH, Ph), 127.8 (CH,Ph), 127.81 (CH, Ph), 127.9 (CH, Ph), 128.3 (CH, Ph), 132.0 (CH, 7-C),132.4 (CH, 7-C), 136.5 (quat., Ph), 155.8 (quat., NCO₂), 156.2 (quat.,NCO₂), 167.2 (quat., 2-C), 167.4 (quat., 2-C), 171.2 (quat., 12-CO) and171.4 (quat., 12-CO); m/z (EI+) 414.2154 (M⁺. C₂₃H₃₀N₂O₅ requires414.2154).

(9R,12S)-1,4-Diaza-4-tert-butyloxycarbonyl-2-oxobicyclo[7.3.0]dodec-2-carboxylicacid 66

To a stirred solution of alkene 65 (0.12 g, 0.29 mmol) intetrahydrofuran (4 cm³) was added platinum oxide (0.0066 g, 0.029 mmol)under a flow of nitrogen. The mixture was hydrogenated (1 atm of H₂) for16 h, filtered through Celite™, and the solvent removed in vacuo. Theresidue was dissolved in a solution of hydrobromic acid in acetic acid(30% w/w, 3 cm³) and stirred at room temperature for 2 h. Removal of thevolatiles in vacuo at 30° C. followed by addition and evaporation ofmethanol:water (3:1) several times yielded the hydrobromide salt whichwas dissolved in a solution of saturated sodium hydrogen carbonate (3cm³). Dioxane (2 cm³) was then added and di-tert-butyl dicarbonate addedto the opaque solution (0.076 g, 0.350 mmol). The resultant suspensionwas stirred for 24 h, di-tert-butyl dicarbonate (0.076 g, 0.3504 mmol),added and stirring continued for a further 72 h. Water (10 cm³) wasadded, the solution washed with dichloromethane, and the aqueous layeracidified with 2 M aqueous hydrochloric acid and extracted withdichloromethane. The combined organic layers were dried Na₂SO₄),filtered and the solvent removed to yield an oil (0.08 g) that waspurified by chromatography (SiO₂, 2:1:0, 2:1:0.3, hexanes-ethylacetate-acetic acid) to give acid 66 (0.074 g, ca. 78%) as a colourlessoil. Acid 66 existed exclusively as the trans C(O)—NPro conformer. Inaddition, restricted rotation about the N—CO carbamate bond was alsoobserved resulting in a 1:1 mixture of conformers: 4 (400 MHz; CDCl₃;Me₄Si) 1.25-1.48 [11H, m, C(CH₃)₃, 8-H_(A)H_(B) and 6-H_(A)H_(B) or7-H_(A)H_(B)), 1.54-1.82 (5H, m, 8-H_(A)H_(B), 10-H_(A)H_(B) and6-H_(A)H_(B) or 7-H_(A)H_(B)), 1.90-2.03 (1H, m, 10-H_(A)H_(B)),2.10-2.33 (2H, m, 11-H₂), 2.78 (1H, td, J 12.7 and 2.6, 5-H_(A)H_(B)),3.78-3.95 (2H, m, 5-H_(A)H_(B) and 3-H), 4.20-4.30 (1H, m, 9-H), 4.52(0.5H, d, J 17.7, 3-H_(A)H_(B)), 4.62 (1H, t, J 8.6, 12-H) and 4.76(0.5H, d, J 17.2, 3-H_(A)H_(B)); δ_(C) (100 MHz; CDCl₃) 22.2 (CH₂, 6-Cor 7-C), 27.6 (CH₂, 6-C or 7-C), 24.3 (CH₂, 11-C), 24.8 (CH₂, 11-C),26.8 (CH₂, 6-C or 7-C), 27.9 (CH₂, 6-C or 7-C), 28.3 [CH₃, C(CH₃)₃],32.6 (CH₂, 10-C), 33.1 (CH₂, 10-C), 34.8 (CH₂, 8-C), 35.3 (CH₂, 8-C),51.1 (CH₂, 5-C), 51.7 (CH₂, 5-C), 54.7 (CH₂, 3-C), 55.9 (CH₂, 3-C), 56.6(CH, 9-C), 56.7 (CH, 9-C), 60.8 (CH, 12-C), 61.1 (CH, 12-C), 80.7[quat., C(CH₃)₃], 154.8 (quat., NCO₂), 155.6 (quat., NCO₂), 170.0(quat., 2-C), 170.5 (quat., 2-C), 174.1 (quat., 12-CO) and 174.7 (quat.,12-CO); m/z (FAB+) 327.1925 (MH⁺. C₁₆H₂₇N₂O₅ requires 327.1920).

(2S,9′S,12′S)-Di-tert-butyl2-{[(1′,4′-diaza-4′-tert-butyloxycarbonyl-2′-oxobicyclo[7.3.0]dodecyl)-12′-carbonyl]amino}-1,5-pentanoate67

Bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BoP—Cl) (0.08 g, 0.31mmol) was added to a solution of acid 66 (0.07 g, 0.20 mmol), L-glutamicacid di-tert-butyl ester hydrochloride 21 (0.085 g, 0.289 mmol) andtriethylamine (0.084 cm³, 0.60 mmol) in dichloromethane (7 cm³) at 0° C.The mixture was stirred for 24 h, washed with saturated aqueous sodiumhydrogen carbonate, 2 M aqueous hydrochloric acid, dried (Na₂SO₄),filtered and the solvent removed under reduced pressure. The resultantoil (0.141 g) was purified by chromatography (SiO₂, 1:1, 1:2,hexane-ethyl acetate) to afford amide 67 (0.077 g, ca. 64%) as acolourless oil. Amide 67 existed exclusively as the trans C(O)—NProconformer. In addition, restricted rotation about the N—CO carbamatebond was also observed resulting in a 46:54 mixture of conformers: δ_(H)(300 MHz; CDCl₃; Me₄Si) 1.14-1.30 [2H, m, 8′-H_(A)H_(B) and H′-6(1H) orH′-7(1H)], 1.34-1.41 [27H, m, 3×C(CH₃)₃], 1.54-2.28 (11H, m,8′-H_(A)H_(B), 10′-H₂, 3-H₂, 4-H₂, 6′-H(3H) or 7′-H(3H)] and11′-H_(A)H_(B)), 2.31-2.49 (1H, m, 11′-H_(A)H_(B)), 2.60-2.70 (1H, m,5′-H_(A)H_(B)), 3.65-3.87 (2H, m, 5′-H_(A)H_(B) and 3′-H_(A)H_(B)), 4.18(1H, br s, 9′-H), 4.39 (1H, q, J 7.6, 2-H), 4.48 (0.5H, d, J 17.5,3′-H_(A)H_(B)), 4.65 (1H, t, J 7.8, 12′-H), 4.74 (0.5H, d, J 17.0,3′-H_(A)H_(B)), 7.45 (0.46H, d, J 7.4, N—H), and 7.64 (0.54H, d, J 7.7,N—H); δ_(C) (75 MHz; CDCl₃) 22.7 (CH₂, 11′-C), 23.3 (CH₂, 6′-C or 7′-C),23.4 (CH₂, 6′-C or 7′-C), 27.0 (CH₂, 3-C, 6′-C or 7′-C), 27.7 (CH₂, 3-C,6′-C or 7′-C), 27.8 (CH₂, 3-C, 6′-C or 7′-C), 28.2 (CH₂, 3-C, 6′-C or7′-C), 27.9 [CH₃, C(CH₃)₃], 28.0 [CH₃, C(CH₃)₃], 28.4 [CH₃, C(CH₃)₃],31.4 (CH₂, 4-C), 32.5 (CH₂, 10′-C), 32.8 (CH₂, 10′-C), 34.4 (CH₂, 8′-C,35.1 (CH₂, 8′-C), 50.7 (CH₂, 5′-C), 51.5 (CH₂, 5′-C), 52.25 (CH, 2-C),52.33 (CH, 2-C), 54.6 (CH₂, 3′-C), 55.0 (CH₂, 3′-C), 56.6 (CH, 9′-C),56.8 (CH, 9′-C), 60.2 (CH, 12′-C), 60.8 (CH, 12′-C), 80.4 [quat.,C(CH₃)₃], 80.6 [quat., C(CH₃)₃], 81.9 [quat., C(CH₃)₃], 154.7 (quat.,NCO₂), 155.5 (quat., NCO₂), 169.4 (quat., 2′-C), 169.8 (quat., 2′-C),170.6 (quat., 12′-CO, 1-C, 5-C), 170.8 (quat., 12′-CO, 1-C, 5-C) and171.8 (quat., 12′-CO, 1-C, 5-C); m/z (FAB+) 568.3592 (MH⁺. C₂₉H₅₀N₃O₈requires 568.3598).

(2S,9′R,12′S)-2-{[(1′,4′-Diaza-2′-oxobicyclo[7.3.0]dodecyl)-12′-carbonyl]amino}-1,5-pentanedioicacid trifluoroacetate 68

Amide 67 (0.072 g, 0.126 mmol) was dissolved in a mixture ofdichloromethane and trifluoroacetic acid (5:2, v/v) and stirred at roomtemperature for 4 h. Evaporation of the volatiles, and subsequentpurification by RP-HPLC [water (0.05% trifluoroaceticacid):acetonitrile, 90:10, 13 ml min⁻¹], gave 68 (0.059 g, 80%) as awhite solid after drying on a freeze drier. Macrocycle 68 existedexclusively as the trans C(O)—NPro conformer: mp^(τ) 30-130° C.; [α]_(D)−36.9 (c 0.195 in MeOH); δ_(H) (400 MHz; CDCl₃; Me₄Si) 1.71-2.29 (11H,m, 6′-H₂, 7′-H₂, 8′-H₂, 10′-H₂, 3-H₂ and 11′-H_(A)H_(B)), 2.33-2.39 (1H,m, 11′-H_(A)H_(B)), 3.27 (1H, dt, J 10.2 and 4.1, 5′-H_(A)H_(B)), 3.39(1H, td, J 12.0 and 2.2, 5′-H_(A)H_(B)), 3.76 (1H, d, J 13.6,3′-H_(A)H_(B)), 4.25-4.20 (2H, m, 3′-H_(A)H_(B) and 9′-H), 4.45 (1H, dd,J 9.3 and 5.1, 2-H) and 4.51 (1H, dd, J 9.2 and 8.3, 12′-H); δ_(C) (100MHz; CDCl₃) 24.06 (CH₂, 6′-C or 7′-C), 24.1 (CH₂, 6′-C or 7′-C), 25.7(CH₂, 3-C), 27.7 (CH₂, 11′-C), 29.6 (CH₂, 4-C), 32.5 (CH₂, 10′-C), 33.4(CH₂, 8′-C), 43.3 (CH₂, 5′-C), 46.8 (CH₂, 3′-C), 51.8 (CH, 2-C), 61.9(CH, 12′-C), 62.8 (CH, 9′-C), 116.2 (quat., q, J 290, CF₃), 162.6(quat., q, J 35.2, CF₃CO₂H), 164.7 (quat., 2′-C), 173.4 (quat., 12′-CO),174.6 (quat., 1-C), and 176.8 (quat., 5-C); m/z (FAB+) 356.1823 [MH(freebase)⁺. C₁₆H₂₆N₃O₆ requires 356.1822].

Example 7 Synthesis of(2S,3′S,8′S,11′S)-2-{[(3′-Amino-1′-aza-2′-oxo-bicyclo[6.3.0]undecyl)-1′-carbonyl]amino}-1,5-pentanedioicacid trifluoroacetate salt

(2S,5S)-N-tert-Butoxycarbonyl-α-allylglycine-5-allyl-L-prolinetert-butyl ester 69

N,N′-Dicyclohexylcarbodiimide (0.057 g, 0.2761 mmol) was added to astirred solution of amine 14 (0.053 g, 0.251 mmol) andN-tert-butoxycarbonyl-α-allylglycine 2 (0.059 g, 0.2761 mmol) indichloromethane (5 cm³) and the mixture stirred overnight, then filteredthrough Celite™ to remove DCU. The reaction mixture was subsequentlywashed with saturated aqueous sodium hydrogen carbonate, 2M aqueoushydrochloric acid, dried (MgSO₄), filtered and the solvent removed toyield an oil (0.106 g) which was purified by chromatography (silica gel,hexane:ethyl acetate, 7:1, 5:1) to afford diene 69 (0.083 g, 81%) as acolourless oil. Due to the complexity of the NMR spectra, the data isnot reported; m/z (FAB+) 409.2707 (MH⁺. C₂₂H₃₇N₂O₅ requires 409.2702).

(8S,11S)-1-Aza-2-oxo-3-tert-butoxycarbonylamino-11-tert-butoxycarbonylbicyclo[6.3.0]undec-5-ene70

To a degassed solution of diene 69 (0.053 g, 0.129 mmol) in drydichloromethane (32 cm³) was addedbis(tricyclohexylphosphine)benzylidieneruthenium dichloride (Grubbs'catalyst) (0.011 g, 0.0129 mmol) under a flow of nitrogen and theresultant purple solution heated at reflux under a nitrogen atmospherefor 24 h. Further bis(tricyclohexylphosphine)benzylidienerutheniumdichloride (Grubbs' catalyst) (0.011 g, 0.0129 mmol) was added andrefluxing continued for a further 48 h after which time another portionof Grubbs catalyst (0.011 g, 0.0129 mmol) was added and the solutionrefluxed for an additional 24 h. The orange/brown solution was cooled toroom temperature, dimethyl sulphoxide (0.151 cm³, 1.935 mmol) was addedand the solution stirred overnight. The solvent was removed in vacuo andthe residue purified by chromatography (silica gel, hexanes:ethylacetate, 4:3, 3:1, 2:1) to give alkene 70 (0.0237 g, 39%) as anoff-white solid. Alkene 70 was shown to be exclusively trans C(O)—NProconformer: mp 175-195° C.; [α]_(D) −24.9 (c 0.237 in MeOH); δ_(H) (400MHz; CDCl₃; Me₄Si) 1.45 [9H, s, C(CH₃)₃], 1.47 [9H, s, C(CH₃)₃],1.68-1.74 (1H, m, 9-H_(A)H_(B)), 1.82 (1H, td, J 11.0 and 5.9,10-H_(A)H_(B)), 2.17-2.26 (2H, m, 9-H_(A)H_(B) and 10-H_(A)H_(B)),2.27-2.40 (1H, br m, 7-H_(A)H_(B)), 2.49 (1H, br d, J 17.6,7-H_(A)H_(B)), 2.55-2.63 (1H, m, 4-H_(A)H_(B)), 2.66-2.80 (1H, m,4-H_(A)H_(B)), 4.40 (1H, dd, J 8.6 and 4.2, 11-H), 4.45-4.62 (2H, m, 3-Hand 8-H), 5.62-5.68 (2H, m, H-6 and N—H) and 5.80-5.90 (1H, m, 5-H);δ_(C) (100 MHz; CDCl₃) 25.5 (CH₂, 10-C), 27.9 [CH₃, C(CH₃)₃], 28.3 [CH₃,C(CH₃)₃], 32.6 (CH₂, 4-C), 33.4 (CH₂, 7-C and 9-C), 55.8 (CH, 3-C), 57.2(CH, 8-C), 61.5 (CH, 11-C), 79.5 [quat., C(CH₃)₃], 81.2 [quat.,C(CH₃)₃], 128.6 (CH, 5-C and 6-C), 155.1 (quat., NCO₂), 169.6 (quat.,2-C) and 171.2 (quat., 11-CO); m/z (EI+) 380.2304 (M⁺. C₂₀H₃₂N₂O₅requires 380.2311).

(2S,8′S,11′S)-Di-tert-butyl2-{[(1-Aza-2-oxo-3-tert-butoxycarbonylaminobicycio[6.3.0]undec-5-ene)-11-carbonyl]amino}-1,5-pentanedioate73

Alkene 70 (0.021 g, 0.055 mmol) was stirred at room temperature indichloromethane:trifiuoroacetic acid (3:1, v/v, 4 cm³) for 5 h. Removalof the volatiles in vacuo yielded an oil that was dissolved in saturatedaqueous sodium hydrogen carbonate (1.5 cm³). Dioxane (1 cm³) was addedfollowed by di-tert-butyl dicarbonate (0.015 g, 0.066 mmol) and themilky suspension was stirred at room temperature for 21 h. The reactionwas diluted with water until a solution was obtained and extracted withdichloromethane. The aqueous layer was acidified with 2M aqueoushydrochloric acid, extracted with dichloromethane and the combinedorganic extracts were dried Na₂SO₄). Removal of the solvent in vacuogave acid 71 (0.012 g) that was dissolved in dichloromethane (3 cm³).Glutamic acid di-tert-butyl ester hydrochloride 72 (0.013 g, 0.044 mmol)was added and the solution cooled to 0° C. Triethylamine (0.013 cm³,0.092 mmol) and bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BoP—Cl)(0.012 g, 0.048 mmol) were added and the mixture stirred for 19 h. Thereaction mixture was washed with saturated aqueous sodium hydrogencarbonate, 2M aqueous hydrochloric acid, dried (Na₂SO₄) and the solventremoved to yield an oil (0.033 g) which was purified by chromatography(silica gel, hexane:ethyl acetate, 2:1, 1:1, 1:2) to afford amide 73(0.0125 g, 60%, 3 steps) as a colourless oil: [α]_(D) +12.1 (c 0.247 inCH₂Cl₂); δ_(H) (400 MHz; CDCl₃; Me₄Si) 1.45-1.48 [27H, S, 3×C(CH₃)₃],1.65-1.75 (1H, m, 9′-H_(A)H_(B)), 1.87-1.96 (1H, m, 3-H_(A)H_(B)),2.02-2.41 (7H, m, 10′-H₂, 4-H₂, 3-H_(A)H_(B), 9′-H_(A)H_(B) and7′-H_(A)H_(B)), 2.51 (1H, br d, J 17.4, 7′-H_(A)H_(B)), 2.57-2.64 (1H,m, 4′-H_(A)H_(B)), 2.70-2.80 (1H, m, 4′-H_(A)H_(B)), 4.39-4.48 (2H, m,11′-H and 2-H), 4.52-4.62 (2H, m, 8′-H and 3′-H), 5.63-5.69 (1H, m,6′-H, 5.78-5.84 (1H, m, 5′-H), 5.91 (1H, br s, N—H) and 6.73 (1H, d, J7.0, N—H); δ_(C) (100 MHz; CDCl₃) 25.4 (CH₂, 10′-C), 27.2 (CH₂, 3-C),27.9 [CH₃, C(CH₃)₃], 28.0 [CH₃, C(CH₃)₃], 28.3 [CH₃, C(CH₃)₃], 31.3(CH₂, 4-C), 32.5 (CH₂, 5′-C), 33.3 (CH₂, 9′-C.), 33.7 (CH₂, 7′-C), 52.3(CH, 2-C), 56.3 (CH, 3′-C), 57.8 (CH, 8′-C), 61.9 (CH, 11′-C), 79.7[quat., C(CH₃)₃], 80.6 [quat., C(CH₃)₃], 82.0 [quat., C(CH₃)₃], 127.9(CH, 5′-C), 128.6 (CH, 6′-C), 155.4 (quat., NCO₂), 170.4 (quat., 2′-C),170.8 (quat., CO), 171.7 (quat., CO) and 172.6 (quat., CO);

(2S,3′S,8′S,11′S)-2-{[(3′-Amino-1′-aza-2′-oxo-bicyclo[6.3.0]undecyl)-11′-carbonyl]amino}-1,5-pentanedioicacid trifluoroacetate salt 74

PtO₂ (0.001 g, 0.0042 mmol) was added to a stirred solution of amide 73(0.012 g, 0.021 mmol) in ethyl acetate:trifluoroacetic acid (5:3, v/v, 8cm³) under a nitrogen atmosphere. The mixture was hydrogenated (1 atm.of hydrogen) overnight, filtered through Celite™, and the solventremoved in vacuo. NMR and HPLC analysis showed the reaction to beincomplete (both double bond and protecting groups remaining). Thus PtO₂(0.0003 g, 0.0126 mmol) was added to a stirred solution of the residuein tetrahydrofuran:methanol (1:1, v/v, 2 cm³) under a nitrogenatmosphere. The mixture was hydrogenated (1 atm. of hydrogen) overnight,filtered through Celite™, and the solvent removed in vacuo. The residuewas dissolved in dichloromethane (3 cm³), trifluoroacetic acid (1 cm³)added and the solution stirred for 4 h at room temperature. Removal ofthe volatiles in vacuo, purification by RP HPLC [90% water (containing0.05% trifluoroacetic acid):10% acetonitrile, 13 ml min⁻¹] and drying ona freeze drier gave 74 (1.4 mg 12%, from 73) as a thin film. Due to thesmall amount of sample obtained an optical rotation was not recorded:δ_(H) (400 MHz; D₂O) 1.74-2.42 (14H, m, 5′-H₂, 6′-H₂, 4′-H₂, 9′-H₂,3-H₂, 7′-H₂ and 10′-H₂), 2.52 (2H, t, J 8.8, 4-H₂), 4.3-4.4 (1H, m, 2H),4.43-4.5 (1H, m) and 4.62-4.69 (1H, m); δ_(C) (100 MHz; D₂O) 22.3 (CH₂),25.4 (CH₂), 28.4 (CH₂), 29.0 (CH₂), 30.2 (CH₂), 32.4 (CH₂), 33.7 (CH₂),53.9 (CH), 55.1 (CH), 60.2 (CH), 62.8 (CH), 168.1 (quat., CO) and 173.7(quat., CO); Note: due to small amount of sample 2 quaternary carbonsand trifluoroacetate signals not detected; m/z (FAB+) 356.1816 [MH(freebase)⁺. C₁₆H₂₆N₃O₆ requires 356.1822].

In Vitro Testing

The following Examples are provided to demonstrate features of thisinvention. They are not intended to be limiting, and other compositionsand methods of this invention can be developed without undueexperimentation. All of those compositions and methods are considered tobe part of this invention. All references cited herein are incorporatedfully by reference.

All the following experiments were carried out using protocols developedunder guidelines approved by the University of Auckland Animal EthicsCommittee.

Example 8 Effects of Cyclic G-2AllylP on Cerebellar Cell Explants

To determine the effects of the compounds of the invention on neuronalcells in vitro, a series of studies was carried out using cerebellarexplants from adult rats. In vitro systems are suitable for studyingneuronal proliferation, neurite growth, formation of nerve bundles andeffects of toxins on neural cells, effects that parallel effectsobserved in vivo. Thus, results of studies using in vitro cerebellarexplants are predictive of effects of interventions in vivo.

In a first series of studies, effects of glutamate on cerebellarexplants were determined. At physiological concentrations, glutamate isa neurotransmitter in the CNS of mammals, including humans. However, atsufficiently high concentrations, glutamate is neurotoxic, resulting inneuronal cell death. Because glutamate is a naturally occurringneurotransmitter in the CNS of mammals, including humans, and becauseglutamate neurotoxicity is recognized in the art as reflective ofneurotoxicity in general, and including cell death and degeneration, itis a valuable tool useful for identifying and characterizing agentseffective in treatment of neurodegeneration and neural cell death.

Materials and Methods

Cover slips were placed into a large Petri dish and washed in 70%alcohol for 5 minutes, then washed with Millipore H₂O. The cover slipswere air dried, and coated with Poly-D-Lysine (1 mg/ml stock solution inPBS, 90-100 μl) for 2 hours at 34° C.

Extraction of Cerebellar Tissue

Postnatal day 8 Wistar rats were used for the study. The rats weresacrificed and placed in ice for 1 minute, decapitated and thecerebellum removed and placed on ice. Cerebellum tissue was placed in 1ml of 0.65% glucose-supplemented PBS (10 μl 65% stock D (+)glucose/1 mlPBS) in a large Petri dish, chopped up into smaller sections andtriturated with a 1 ml insulin syringe via a 23 G (0.4 mm) needle, andthen squirted back into the glucose solution in the large Petri dish.The tissue was sieved through (125 μm pore size gauze) and centrifuged(2 minutes at 60 g) twice to exchange the medium into serum-freeBSA-supplemented START V medium (Biochrom, Germany). The secondcentrifugation step was done with 1 ml of START V medium. Themicroexplants were reconstituted into 500 μl of START V medium and puton ice.

Cultivation of Cerebellar Cells

Two hours after PDL-coating, the slides were washed with Millipore H₂Oand air dried. Each slide was placed into a small Petri dish (diameter:35 mm) and 40 μl of START V/cell suspension was added. The tissue wasincubated for 2 hours at 34° C. (settlement period). START V-medium (1ml) was then added to the Petri dish and cultivated at 34° C. in thepresence of 5% CO₂ in air at 100% humidity for 48 hours.

Drug Application

For the study, certain explant cultures were exposed to vehicle (PBS)only. In the first study (Study 1) 10 μl of toxin 1 (L-glutamate-100 mMin Millipore water; final concentration: 1 mM) and 10 μl of toxin 2(3-nitropropionic acid-50 mM-pH 7-in Millipore water, finalconcentration: 0.5 mM) was applied simultaneously with the compounds tobe tested (10 mM stock solution prepared in PBS and diluted to finalconcentrations between 1-100 nM). In each case, the drugs were left incontact with the explants for the duration of the study.

Methods for Determining Drug Effects

After explants were exposed to drugs for the study period, cells werethen rinsed in PBS and then fixed in increasing concentrations ofparaformaldehyde (500 μl of 0.4% PFA was applied; then 1.2% PFA; then 3%PFA and finally 4% PFA (each fixation step: 2-3 minutes). Finally, themicroexplants were rinsed in PBS.

Neurons in the explants were then evaluated for morphology (presence ofneurites) and counted as live cells per microscopic field. Four fieldsdisplaying highest cell density were counted per cover slip and the datapresented as mean±standard error of the mean (SEM); n=4 each.Statistical significance was evaluated by using the non-paired Student'st-test.

Results

The results of the study are shown in FIG. 1 and FIG. 2. Glutamatetreatment (1 mM) resulted in about an 80% loss of cerebellar neuronshaving neurites compared to vehicle-treated controls. In contrast,administration of (2S,3′S,8′R,11′S)2-{[(3′-Amino-1′-aza-2′-oxobicyclo[6.3.0]-undecyl)-11′-carbonyl]amino}-1,5-pentanedioicacid trifluoroacetate salt 48 and(2S,9′R,12′S)-2-{[(1′,4′-Diaza-2′-oxobicyclo[7.3.0]dodecyl)-12′-carbonyl]amino}-1,5-pentantedioicacid trifluoroacetate 68 (FIGS. 1 and 2 respectively) significantlyincreased the numbers of cells having neurites in a dose-dependentmanner when administered simultaneously with glutamate. Treatment with(2S,3′S,8′R,11′S)2-{[(3′-Amino-1′-aza-2′-oxobicyclo[6.3.0]-undecyl)-11′-carbonyl]amino}-1,5-pentanedioicacid trifluoroacetate salt, 48 and(2S,9′R,12′S)-2-{[(1′,4′-Diaza-2′-oxobicyclo[7.3.0]dodecyl)-12′-carbonyl]amino}-1,5-pentanedioicacid trifluoroacetate 68 (FIGS. 1 and 2 respectively) showed asignificant recovery from glutamate-induced neurotoxicity.

Conclusions

Treatment with (2S,3′S,8′R,11′S)2-{[(3′-Amino-1′-aza-2′-oxobicyclo[6.3.0]-undecyl)-11′-carbonyl]amino}-1,5-pentanedioicacid trifluoroacetate salt 48 and(2S,9′R,12′S)-2-{[(1′,4′-Diaza-2′-oxobicyclo[7.3.0]dodecyl)-12′-carbonyl]amino}-1,5-pentanedioicacid trifluoroacetate 68 prevented glutamate-induced neurotoxicity,indicating that (2S,3′S,8′R,11′S)2-{[(3′-Amino-1′-aza-2′-oxobicyclo[6.3.0]-undecyl)-11′-carbonyl]amino}-1,5-pentanedioicacid trifluoroacetate salt 48 and(2S,9′R,12′S)-2-{[(1′,4′-Diaza-2′-oxobicyclo[7.3.0]dodecyl)-12′-carbonyl]amino}-1,5-pentanedioicacid trifluoroacetate 68 are neuroprotective and can be used to reverseand/or inhibit neuronal degeneration or cell death.

Example 9 Hypoxic-Ischemic Injury

Materials and Methods

To determine whether compounds of the invention might prevent neuronalinjury in response to stroke, cardiac arterial bypass graft surgery(CABG) or other hypoxic insults, a series of studies were carried out inrats that had been exposed to hypoxic-ischemic injury (HI). Adult rats(Wistar, 280-310 g, male) were used. The modified Levine modelpreparation and experimental procedures were used (Rice et al, 1981,Ann. Neurol.:9: 131-141; Guan et al J., 1993, Cereb. Blood Flow Metab.:13(4): 609-16). These procedures in brief, consist of an HI injuryinduced by unilateral carotid artery ligation followed by inhalationalasphyxia in the animals with an implanted lateral ventricular cannula. Aguide cannula was stereotaxically placed on the top of the dura 1.5 mmto the right of the mid-line and 7.5 mm anterior to the interaural zeroplane under halothane anaesthesia. The right carotid artery was doubleligated two days after the cannulation. After 1 hour recovery from theanaesthesia, each of the rats were placed in an incubator where thehumidity (90±5%) and temperature (31°±0.5° C.) were controlled foranother hour, then exposed to hypoxia (6% oxygen) for 10 min. Theanimals were kept in the incubator for an additional 2 hours beforetreatment.

Pairs of rats were treated intracerebral ventricularly (icv) with either(2S,9′R,12′S)-2-{[(1′,4′-Diaza-2′-oxobicyclo[7.3.0]dodecyl)-12′-carbonyl]amino}-1,5-pentanedioicacid trifluoroacetate (68) (100 pM) or vehicle (normal saline) 2 hoursafter hypoxic-ischemic insult. Rats in each group were simultaneouslyinfused with(2S,9′R,12′S)-2-{[(1′,4′-Diaza-2′-oxobicyclo[7.3.0]dodecyl)-12′-carbonyl]amino}-1,5-pentanedioicacid trifluoroacetate (68) or vehicle under light anaesthesia (1.5%halothane) 2 hours after the insult. A total volume of 20 μl was infused(icv) over 20 minutes by a micro-infusion pump.

Histological examination was performed on rats 5 days after thehypoxic-ischemic injury. The rats were killed with an overdose of sodiumpentobarbital and were perfused transcardially with normal salinefollowed by 10% formalin. The brains were kept in the same fixative fora minimum of 2 days before being processed using a standard paraffinimbedding procedure.

Coronal sections 8 μm in thickness were cut from the striatum, cerebralcortex and hippocampus and were stained with thionin and acid fuchsin.The histological outcome was assessed at three levels: (1) the mid levelof the striatum, (2) where the completed hippocampus first appeared and(3) the level where the ventral horn of the hippocampus just appears.The severity of tissue damage was scored in the striatum, cortex and theCA1-2, CA3, CA4 and dentate gyrus of the hippocampus. Tissue damage wasidentified as neuronal loss (acidophilic (red) cytoplasm and contractednuclei), pan-necrosis and cellular reactions. Tissue damage was scoredusing the following scoring system: 0: tissue showed no tissue damage,1: <5% tissue was damaged, 2: <50% tissue was damaged, 3: >50% tissuewas damaged and 4: >95% tissue was damaged.

Results and Conclusion

The results of this study are shown in FIG. 3. FIG. 3 shows thathypoxic-ischemic injury (left bars of each set) resulted in significantdamage scores in each of the areas of the brain studied. FIG. 3 alsoshows that central administration of a relatively low dose of(2S,9′R,12′S)-2-{[(1′,4′-Diaza-2′-oxobicyclo[7.3.0]dodecyl)-12′-carbonyl]amino}-1,5-pentanedioicacid trifluoroacetate (68) (right bars of each set; 100 pM)significantly reduced the tissue damage in each brain region examinedcompared to the vehicle treated group (p<0.001).

It can be seen that(2S,9′R,12′S)-2-{[(1′,4′-Diaza-2′-oxobicyclo[7.3.0]dodecyl)-12′-carbonyl]amino}-1,5-pentanedioicacid trifluoroacetate (68) can be neuroprotective against neural damagecaused by hypoxic-ischemic injury, even when administered afterhypoxic-ischemic injury. This surprising finding indicates that(2S,9′R,12′S)-2-{[(1′,4′-Diaza-2′-oxobicyclo[7.3.0]dodecyl)-12′-carbonyl]amino}-1,5-pentanedioicacid trifluoroacetate (68) is a useful agent to treat a variety ofconditions characterized by neural degeneration or cell death.

1. A compound having the formula:

or a pharmaceutically acceptable salt thereof, where R¹═—COOH; R²═—(CH₂)₂—COOH; R³═H; X═—(CH₂)₃—.
 2. A compound having the formula:

where the compound is (2S, 3′S, 8′R, 11′S) 2-{[(3′-Amino-1′-aza-2′-oxobicyclo[6.3.0]-undecyl)-11′-carbonyl]amino}-1,5-pentanedioic acid, or a pharmaceutically acceptable salt thereof.
 3. The compound of claim 2, where said salt of said compound is (2S, 3′S, 8′R, 11′S) 2-{[(3′-Amino-1′-aza-2′-oxobicyclo[6.3.0]-undecyl)-11′-carbonyl]amino}-1,5-pentanedioic acid trifluoroacetate.
 4. A compound having the formula:

where the compound is (2S, 3′S, 8′S, 11′S)-2-{[3′-Amino-1′-aza-2′-oxobicyclo[6.3.0]-undecyl)-11′-carbonyl]-amino}-1,5-pentanedioic acid, or a pharmaceutically acceptable salt thereof.
 5. The compound of claim 4, where said salt of said compound is (2S, 3′S, 8′S, 11′S)-2-{[3′-Amino-1′-aza-2′-oxobicyclo[6.3.0]-undecyl)-11-carbonyl]-amino}-1,5-pentanedioic acid trifluoroacetate.
 6. A method of treating a disease selected from the group consisting of ischemic injury, hypoxic injury, reperfusion injury, and asphyxia, comprising administering to a patient an effective amount of a compound of claim
 2. 7. The method of claim 6, further comprising administering another agent selected from the group consisting of insulin-like growth factor-I, insulin-like growth factor-II, transforming growth factor-β1, activin, growth hormone, nerve growth factor, growth hormone binding protein, IGF-binding protein, basic fibroblast growth factor, acidic fibroblast growth factor, the hst/Kfgk gene product, FGF-3, FGF-4, FGF-6, keratinocyte growth factor, androgen-induced growth factor, int-2, fibroblast growth factor homologous factor-1 (FHF-1), FHF-2, FHF-3 and FHF-4, keratinocyte growth factor 2, glial-activating factor, FGF-10 and FGF-16, ciliary neurotrophic factor, brain derived growth factor, neurotrophin 3, neurotrophin 4, bone morphogenetic protein 2, glial-cell line derived neurotrophic factor, activity-dependant neurotrophic factor, cytokine leukaemia inhibiting factor, oncostatin M, an interleukin, α-interferon, β-interferon, γ-interferon, consensus interferon, TNF-α, clomethiazole; kynurenic acid, Semax, tacrolimus, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, adrenocorticotropin-(4-9) analogue [ORG 2766], dizolcipine [MK-801], selegiline, a glutamate antagonist selected from the group consisting of NPS1506, GV1505260, MK-801 and GV150526, an AMPA antagonist selected from the group consisting of 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), LY303070 and LY300164 and an anti-inflammatory agent selected from the group consisting of anti-MAdCAM-1 antibody and an antibody against an integrin α4β1 receptor and an integrin α4β7 receptor.
 8. The method of claim 7 wherein said anti-MAdCAM-1 antibody is MECA-367.
 9. The method of claim 6, in which the amount is in the range of about 0.00 1 mg/kg to about 100 mg/kg mass of said mammal.
 10. The method of claim 6, wherein said administration is via the cerebrospinal fluid and said amount is in the range of about 0.001 mg/kg to about 0.1 mg/kg mass of said mammal.
 11. The method of claim 6, wherein said administration is via oral, systemic or parenteral route and said amount is in the range of about 1 mg/kg to about 100 mg/kg mass of said mammal.
 12. The method of claim 6, wherein said mammal is a human being.
 13. The method of claim 6, wherein said compound is administered in a pharmaceutically acceptable solution.
 14. The method of claim 6, wherein said compound is administered into a lateral cerebral ventricle.
 15. The method of claim 6, wherein said compound is administered in a solution having a concentration of compound in the range of about 0.0001% by weight to about 10% by weight. 